Raoul L. Wolf, MD gives a presentation on Immunodeficiency: A Multi-Specialty Collaboration.
[MUSIC PLAYING] DR. RAOUL L. WOLF: The reason for this talk is to try to take a look at the complexities and difficulties of diagnosing and assessing and evaluating immune deficiency. And also what's new and what is really cutting edge in terms of therapy, which Dr. Cunningham is going to talk about in terms of genetic therapy and in terms of transplantation. And so I'm going to introduce to you a young lady who is a three-month-old girl who was born after an uneventful pregnancy. Shortly after birth, she was noted to have cold extremities and was diagnosed after a day-- noted her oxygen saturation had dropped down into the 40s-- that she had an interrupted aortic arch and some other intracardiac defects. After corrective surgery, including a Norwood, she stabilized. And at the time of surgery, the surgeon noted that she did not have a thymus. It was also noted shortly after the surgery, that she developed hypocalcemia and was subsequently diagnosed as having hypoparathyroidism as well. Her only complication was line sepsis with serratia. And she had not had and has not had any other infections since that initial episode. She required prolonged ventilation, intubation following the surgery and the procedures, which inevitability led to tracheomalacia, and she also required a G-tube for feeding. When we saw her for the first time, she was comfortable. She was not cyanosed, and her vital signs and general examination were within normal limits. She had typically rounded, low-set posteriorly rotated ears and a triangular-shaped face. She had a small cob-shaped mouth and remarkably long philtrum. The palate was intact. Her heart showed multiple murmurs, and S1, S2 were normal. Lung fields were clear, and otherwise her physical examination was unremarkable. This is obviously not our patient we're talking about but another patient with a similar problem. This was a young man so many years ago with a less fortunate outcome. What is striking, particularly in the lateral, is the shape of the ears which are rounded. They're posteriorly rotated and clearly low-set. The other striking feature is the triangular shape to the mouth, which even shows post mortem-- to the face, I beg your pardon-- with a small, downturned, cob-shaped mouth. Philtrum is unusually long and the nose has a very beaked appearance. So these are the typical features that are often seen in what is termed to DiGeorge syndrome, and I'll come back to that. The results of studies that were done at the time, showed a relatively interesting immunoglobulin pattern with a normal-to-high IgM and IgG and IgA being acceptable for a three-month old. However, a repeat a month later shows that IgG has not increased nor has IgA. And IgM remains high. This is the sort of pattern one might see where there is a signaling defect, and there is a defect in switch from isotype with the switch from IgM to IgG and IgA. The lymphocyte subsets that were done at the time-- and I think this slide shows very clearly that immunologists have cornered the market on [INAUDIBLE] contractures. However, the results of interest here are predominantly that she is lymphopenic, with low lymphocytes, generally. Has very low level of CD3, general T-cell marker cells, which have declined still further at subsequent testing. And her other numbers, subtype CD4 and CD8, are also in decline and have low numbers. B cells, CD19 cells, are high, which is not surprising. It's a more relative increase. And she does show an increase in CD1A. CD1A is a thymocyte marker. It's a primitive T-cell marker. And this shows some peripheral circulating T-cell markers of primitive thymocytes. These markers indicate activity. And there are activated cells, but an equal number to naive cells. These would be naive cells. Those would be activated cells. But both of those, again, declining in numbers as time has gone by. Looking at lymphocyte proliferation-- I'll cut this one short-- shows normal proliferation. These are measured by use of pokeweed mitogin and phytohemagglutinin. Both of these are plant lectins and are extremely potent antigens which stimulate T-cells very strongly to proliferate. As you see, there's no proliferative response from B cells, which is not surprising. So the general CD3 proliferation is normal for both of these plant lectins. Again, not surprisingly, genetics showed a deletion in the region of 22q11.2, which is the region that has been referred to as DiGeorge syndrome. There's an interesting confusion that has arisen because of the original terms that we use to describe this deletion which occur in truncal abnormalities, [INAUDIBLE] palatine abnormalities, DiGeorge syndrome-- all of which are aspects of the same problem. So there are conotruncal cardiac defects, which we'll look at a little more closely; hypocalcemia, associated with hypoparathyroidism; absence of the thymus; and T-cell defects. And most of this relates to disruption an embryogenesis, predominantly around the third and fourth branchial arches and the second and third branchial pouches. But the whole family of 22q11 syndromes is enormous, so the range of defects is extremely large. The general features are-- as we've described-- the triangular-shaped face; the low-set, rounded, posteriorly rotated ears; the cob-shaped mouth; the short philtrum with a beaked nose; and either bifid uvula-- which is common-- and cleft palate, which is less common. Cardiac defects are mostly conotruncal, in other words outflow tracked. And they are interrupted aortic arch, double aortic arch, transposition VSD, double outlet right ventricle, tetralogy of Fallot, and so on. The immune defects are predominantly T-cell deficiencies but also associated with a paucity of lymphatic tissue. Hypoparathyroidism is the predominant endocrine phenomenon, but growth hormone deficiency has also been associated as has renal abnormalities. What is probably less commonly known is that this defect also affects neural development. And associated with it are learning defects, including attention deficit hyperactivity and a number of other related disorders, as well as seizures. And a number of psychiatric disorders, including schizophrenia, have been associated with this defect as well. And then there are other skeletal abnormalities, including hand abnormalities and arm abnormalities. So how does this all come down to one tiny little chromosome? There are several defects which occur which Dr. Cunningham is going to go into in more detail. But I want to just point to what is probably the main one and the one that predominantly causes the problem. And this is within the 22q11.2 region. In fact, the 22q11.21 region is a chromosome which governs TBX. TBX1 is the Tbox 1 protein. This protein has a tremendous number of controlling effects and is a transcription factor which acts on a number secondary genes, predominantly those involved with fibroblast growth factor; retinoic acid receptor; beta-catenin, which is associated with cell adhesion and is very important for cells sticking together; and bone morphogenic protein, which has obvious effects in bone development. So these effects predominantly occur in the pharyngeal pouches, in the neural crest, and in the developing limb buds is where the predominant targets of these defects lie. So face is involved, giving rise to the defects we talked about. Cardiac defects, the heart develops from the second through fourth branchial arches. The face develops from the branchial pouch. Thymus develops from the second and third branchial pouches, the parathyroids from the same location. And then the limb buds, which initially are cranial and track caudally, are also affected. So predominantly through this Tbox 1 protein is what ties together this collection of defects. And then finally, because of neural crest involvement, one also has brain involvement with many of the defects that we alluded to. So why does this problem with the thymus give rise to immune deficiency? The thymus is a most intriguing organ. It is an organ that absolutely has to be present during embryogenesis and after birth seems to have very little function. It can be removed if it gets in the way of complex cardiac surgery without any obvious immunologic ill effects or subsequent problems. So as we said, the thymus is where T-cells learn the most important lesson the immune system can learn, and that is they're to recognize self major histocompatibility complex, MHC. Without the ability to recognize what is self, the immune system cannot recognize what is non-self and becomes nonfunctional. So what happens in the thymus is a series of development steps in which these lymphoblasts that migrate into the thymus are challenged with a series of MHC antigens. If they react and start dividing, it triggers apoptosis, and the cells self-destruct. About 5% of the lymphoblasts that enter the thymus emerge as functional T-cells, so there is tremendous cell loss in this education process. In the absence of a thymus, there are no functional T-cells, or poorly functional T-cells. The other important point is that in humans, as opposed to rodents, B cells are completely dependent on T-cells for function, and the B cell/T-cell interaction is MHC-restricted, as is the T-cell/macrophage interaction so that the role of MHC is crucial in human immunologic function. So looking then at how this all falls out, T-cell development starts with a pluripotent lymphoblast that then migrates into the developing thymus. In the thymus, it undergoes this education process and emerges as a functional, long-lived T-cell. This is not the only way, though, in which T-cells can arise. The thymus also acts on null or uncommitted cells, lymphocytes, by a series of hormones known as thymosins. The facteur thymique is the best described of these. But these are produced by the Hassall's corpuscles, which are epithelial constructs within the thymus. The action on null cells also forms T-cells which have a different identity and probably a different function. 65% of circulating lymphocytes are T-cells which then migrate into the secondary immune organs-- which are the lymph nodes, bone marrow, spleen, and tonsils. And in the second organs, the T-cells form the cortex surrounding B cells. So looked at another way then in a little more detail within the thymus, what is happening is a series of changes at the T-cell surface. This is occurs in concert with the development of the thymus. So initially, the cells enter the subcapsular epithelium, moving into the cortical epithelium as it develops within the thymus, and finally medullary epithelium. And later, Hassall's corpuscles emerge from the medullary epithelium. So the initial cells to enter the thymus have few markers on them, but they have primitive markers which are CD1, CD2, and CD5. These are all very primitive markers that have little function in adults or mature children. There are two branches that then occur as they move through the thymus. Initially, CD3, CD4-negative, CD8+, and still CD2 and CD5+ and another subset that will be CD8+ and CD25+-- sorry that should be CD4+-- and CD8-negative. Those then combine to a multipotent cell that has CD3 more or less present with CD4+ and CD8-negative. And then becomes totipotent with markers of 3, 4, and 8+. And finally settles down to the division we're familiar with, CD4 and CD8 cells and with CD3 being the universal T-cell receptor. So what happens if you don't have T-cells? Why is this bad for you? Well, the consequence is a severe viral infections with unusual organisms, such as pneumocystis jirovecii, atypical mycobacteria, and other organisms that are normally non-pathogens. In fact, pneumonia with pneumocystis or with cytomegalovirus outside of the newborn period is pathognomonic of dysfunctional T-cells. There are also infections with organisms that are normally weak pathogens, such as pseudomonas [INAUDIBLE]. Fungal infections, again, are predominant, and many malignancies result from having poorly functional T-cells. Also malignancies such as Kaposi's sarcoma that are normally mild, non-invasive malignancies can become invasive. So how does one investigate the potential as in this child? I'm just going to go through what some of these investigations mean. So particularly as a broad opening, investigation one would obviously want a CBC with platelets and differential, see if cells are available, and quantitative immunoglobulins. It's not enough to do quantitative immunoglubulins, one should look at specific antibodies as well. So morphology of cells-- particularly T-cells-- the surface markers, and cell function. The only cell function that one can actually assist is of T-cells. B cells do not proliferate and do not demonstrate function in vitro. So these markers which are commonly seen, the information that they provide is of some cell types and not necessarily strongly correlated with function. For example, the CD5 marker, as we said, is a primitive B cell marker which tends to disappear but later on is associated with the development of tolerance. CD9 is equally a pre-B cell, early B cell marker. CD19 is the common B cell marker, and CD21 and others are in fact complement markers. The B cells are unique in that they have receptors for immunoglobulin and there are Fc portion receptors on the B cells. What is also interesting is that B cells have IgD and IgM on their surface. They do not have IgG. And they do not have IgA. This is the function for IgD, which functions or maintains memory. IgD is the memory immunoglobulin on B cells. The CD4 cells bind with MHC class II and CD8 cells bind with MHC class I. They are neither of them particularly helper nor suppressor cells. They trigger different responses when binding with MHC and with macrophages. The CD56/16 series are natural killer cells of various types which predominantly control viruses and become a serious problem. If T-cells do not develop, as in this case where the thymus is absent. And as we've mentioned, CD1A, CD19, and CD90 and other series are primitive thymocytes and an indicator of a poorly functional thymus. Of some of the more complex compound markers that are available-- for example, the three CD56/16 series that are shown there, depending on various intensities of staining, have different functions. The most common cytotoxic natural killer cells have a weakly staining 56 and a strongly staining 16. Other compound markers that are important, like CD3, 4, and 8 combined with CD25-- CD25 is the alpha chain of the interleukin 2 receptor, which is a key in immunologic conversation and in stimulating immune response. So abnormalities at that level can give rise to nonfunctional immune response. And then just briefly, the overview of management which Dr. Cunningham is going to look at it in more detail is predominantly from a clinical point of view. Avoidance of high risk situations. Treating, obviously, with antibiotics either acutely or in some cases, long-term chronic antibiotic management. Replacement therapy, most commonly with intravenous gamma globulin, but otherwise bone marrow transplantation and gene therapy. And in terms of general management, the avoidance of high risk situations. Nursery schools, obviously a hotbed of infection. Blood products, and even if one is only suspicious, should use only washed, irradiated red blood cells. The last thing you want to do is to infuse viable lymphocytes into a patient whose immune deficient. And finally, live virus vaccines should be avoided.
