Regulatory T Cell involvement with Maternal Fetal Tolerence

Pregnancy is an emotional time for mothers. The constant mood swings, cravings, and nesting syndrome makes the 9 months seem like a lifetime. While the mother is busy attending doctor’s appointments, setting up a nursery and attempting to be ready for a child, her body’s immune system is attempting to reconcile with the fetus growing inside the womb. The immune system sees this new life as a foreign invader attempting to overwhelm the body’s systems (which it kind of is). This is a sign that the immune system should attack and rid the body of the invader. However, if the immune system did this every time a woman got pregnant; our lineage would have ended with Eve. So we as humans have evolved ways to regulate the immune system during pregnancy in order to give birth to health babies. Different stages in pregnancy bring changes in cytokine production (the immune system’s messenger, of which there are many different kinds) to modulate the inflammatory response as needed (1). These changes in cytokine production then produce changes in the cells of the immune system, macrophages (collect debris), natural killer cells (does just what it sounds like it does, kills foreign cells), and regulatory T cells (regulates the other cells in the immune system) (2). Even though these regulatory actions exist there still exists evidence that the maternal immune system can and does respond to fetal antigen (fetal cells).

The presence of maternal antibody (the immune system’s detector) to major histocompatibility complex (MHC) (the complex that presents foreign material) suggests that CD4 T cells can participate in anti-fetal responses (2). A study on the modification of immune cells during pregnancy done by Elizabeth Bonney, examined spleen CD4 T cells of both normal mice and a unique CD4 T cell receptor transgenic mouse specific for the male antigen H-Y (a genetically mutated mouse that cause rejection of fetal material when mated with another H-Y mouse)(3). They found that during pregnancy, these transgenic CD4 T cells become activated and multiply in response to specific antigen and modulate the expression of the CD4 co-receptor (receptor that helps CD4 bind). This data suggest that during pregnancy this particular T cell pool is dynamic and further suggests that there is a specific mechanism of regulation that helps to maintain the coexistence between fetal tolerance and maternal T cell responsiveness.

They first analyzed CD4 T cells in the spleens and uterine draining lymph nodes of normal mice. They found that the proportion of CD4 T cells decreases at mid-gestation. They analyzed the different cells through flow cytometry analysis (separates the cells based on markers expressed on cell surface, in this case CD25 and CD44). In the spleen, the proportion of CD4 T cells expressing CD25 (regulatory T cells) and a low level of CD44 (if the Treg express CD44 it will enhance the immunosuppressive effects of the Treg) remained stable throughout gestation (4). Pregnancy supported an increase in the proportion of CD25 expressing CD4 T cells bearing intermediate levels of CD44. In the uterine nodes the proportion of CD25-positive CD4 T cells increased only transiently in early–mid-gestation, regardless of CD44 level. In the uterine draining nodes, the proportion of CD4 T cells expressing high levels of CD44 did not change during pregnancy. The total number of CD4 T cells in the spleens of pregnant animals was not significantly different from those observed in non-pregnant mice. However, in late gestation there was a significant increase in the number of activated (CD44^high CD25^low) CD4 T cells present during the later third of gestation. This data suggested that pregnancy results in the activation of a subset of CD4 T cells, while the overall size of the CD4 T cell pool is maintained.

There are several possible explanations for the observed maintenance of total cell numbers associated with an increase in cells bearing an activated marker for immunosuppression. These include incomplete activation, rapid activation and multiplication balanced by death, or other subtle regulation of the CD4 T cell pool. To attempt to elucidate this mechanism they studied CD4 T cells in a TCR transgenic mouse specific for the male minor histocompatibility antigen H-Y. The proportion of maternal CD4⁺ Vβ6⁺ T cells (superantigen responsive T-cells) in the spleen decreased significantly during pregnancy, but this population recovered by 1 week after birth (5). In contrast, the proportion of CD4⁺ Vβ6⁺ T cells in the uterine draining nodes was relatively constant throughout gestation. The decrease in splenic CD4⁺ Vβ6⁺ T cells was probably related to an expansion of non-T cells, which was also observed in normal mice.

They then examined the number of CD4⁺ Vβ6⁺ cells. On days 8–10 of gestation, the number of CD4⁺ Vβ6⁺ T cells in the spleen was not statistically different from the number found in unmated mice. However, at gestational age 12 the number of T cells dropped significantly and continued to decrease by day 14. By 4–7 days post-delivery, the number of CD4⁺ Vβ6⁺ spleen T cells returned to normal. To investigate the changes in the CD4⁺ Vβ6⁺ T cell population in the spleen at mid-gestation, they then examined the expression of CD4 and the zeta chain of the TCR complex, both of which have been implicated in T cell signaling. CD4⁺ Vβ6⁺ cells were isolated by flow-sorting from spleens of pregnant mice and three unmated controls. They found that CD4⁺ Vβ6⁺ T cells from mid-gestation pregnant spleen expressed similar levels of CD4 and the zeta chain compared to cells of the same phenotype isolated from the spleens of unmated controls. They then tested the antigen-specific responsiveness (how well these cells will responds to specific antigens) of spleen CD4⁺ Vβ6⁺ T cells. Responsiveness towards the H-Y specific mutation was determined by calculating the ratio of cell proliferation (cell multiplying) of CD4⁺ Vβ6⁺ T cells of pregnant or unmated TCR transgenic mice. On days 8–10 of gestation, male-specific proliferation in pregnant samples was similar to unmated mice, meaning the antigen responses were the same. On days 12–16 of gestation there was a non-significant trend towards increased H-Y-specific responsiveness in pregnant mice.

In order to examine the behavior of CD4 anti H-Y cells in a more normal environment (since the H-Y transgenic were of the same specificity), these cells were transferred into normal mice to generate chimeras (a mouse with two genetically distinct cell populations, makes it easier to detect cell activities and migration) well before pregnancy. Examination of the spleen cells present in either the unmated or pregnant mice revealed cells of both host and donor (anti H-Y) origin. However, there was a decrease in the proportion of donor-derived H-Y-specific T cells expressing CD4 compared to non-transgenic T cells. This suggests that the observed decrease in CD4 expression seen during pregnancy in this population of spleen T cells might be antigen-specific.

This observed decrease in the proportion of transgenic CD4 T cells may be a transition from CD4⁺ Vβ6⁺ to CD4⁻ Vβ6⁻. To investigate this possibility, they examined the spleen cells from pregnant and unmated transgenic mice the presence of possible intermediate cell types. They found that unmated mice had an increased number of CD4⁻ Vβ6⁺ cells in the spleen. They also found that the number of these cells in the spleen was higher in pregnant transgenic mice compared with non-pregnant controls. They then hypothesized that the appearance of CD4⁻ Vβ6⁺ T cells may have been due to a decreased expression of CD4. They analyzed the RNA expression of the zeta chain of the TCR and CD4 and found that the relative expression of the TCR zeta chain was similar in CD4⁺ Vβ6⁺ and CD4⁻ Vβ6⁺. They found a trend towards decreased expression of CD4 in Vβ6⁺ cells that do not express CD4 on the cell surface. However, this did not correlate with increased expression of Runx1, a transcription factor thought to participate in transcriptional down-regulation of CD4.

They hypothesized that one potential mechanism underlying the decreased surface expression of CD4 may be transient internalization of CD4 into the cytoplasm. They analyzed pregnant transgenic mice for intracellular CD4 protein and it was found to be present in both CD4⁺ Vβ6⁺ and CD4⁻ Vβ6⁺ cells. They then put these cells into culture for 36hrs and found that cells that were initially surface-negative for CD4 re-expressed CD4 after culture, while cells positive for CD4 retained surface expression. Therefore down-regulation of CD4 on T cells during pregnancy leaves overall immune responsiveness preserved which is needed to protect the pregnant woman from infections that would threaten both her and the fetus but leaves a potentially higher but modifiable threshold for fetal antigen-specific responses. The researchers then predict that maternal T cells that when activated by fetal antigen down-regulate or die. However, maternal fetal tolerance is still a complex issue that requires increasingly sophisticated models for examination.

Primary Article

1) Bonney, E. A., Shepard, M. T. and Bizargity, P. (2011), Transient modification within a pool of CD4 T cells in the maternal spleen. Immunology, 134: 270–280

Supplementary Articles

1) Mor G, Cardenas I: The immune system in pregnancy: a unique complexity. Am J Reprod Immunol 2010; 63:425–433.

2) Mor G, Cardenas I, Abrahams V, Guller S: Inflammation and pregnancy: the role of the immune system at the implantation site. Ann N Y Acad Sci 2011; 1221:80–87.

3) 3) Simpson, Elizabeth. Review Lecture: Immunology of H-Y Antigen and its Role in Sex Determination. Proceedings of the Royal Society of London. Series B, Biological Sciences , Vol. 220, No. 1218 (Nov. 22, 1983), pp. 31-46

4) 4) Liu T, Soong L, Liu G, Konig R, Chopra AK. CD44 expression positively correlates with Foxp3 expression and suppressive function of CD4+ Treg cells. Biol Direct. 2009;4:40.

5) 5) Luther, S. A., Serre, K., Cunningham, A. F., Khan, M., Acha-Orbea, H., MacLennan, I. C. and Toellner, K. M., Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells. Eur. J. Immunol. 2007. 37: 1476–1484