Danny J. was born a healthy 8 pound baby, the fourth child of Doris and Donald
J. His older brother had died of pneumonia in infancy five years before; his
two older sisters had had only the usual childhood colds and stomach upsets.
Danny's parents brought him to the emergency room when he was about six months
old gasping for breath and with a 104° fever. His lymph nodes were not swollen
and he had previously suffered from only minor respiratory infections. A throat
swab and blood sample were collected for lab analysis. Danny was admitted to
the hospital and treated with broad spectrum antibiotics and oxygen. The throat
culture was positive for Streptococcus pneumoniae, a Gram positive coccus
surrounded by a thick polysaccharide capsule that protects the bacterium from
phagocytosis. The white blood cell count and total plasma protein were slightly
below normal. The infection responded to antibiotics and Danny was able to go
home after a week. Follow-up tests were performed on an outpatient basis.
Serum immunoelectrophoresis revealed the nearly complete absence of antibodies from Danny's serum. IgG was present at about 10% of normal levels, and the other isotypes were not detected. Blood and bone marrow were collected for phenotypic analysis. Circulating lymphocytes were predominantly CD3+ IgM- IgD-; less than 0.1% were IgM+ IgD+, where normally circulating lymphocytes are 5-15% IgM+ IgD+. Granulocytes and monocytes were present in normal numbers. The bone marrow contained cells which were positive by immunofluorescence for membrane μ, λ5-VpreB, Igα and Igβ, but not membrane or cytoplasmic κ, λ, or δ chain or CD19. Bone marrow was positive by PCR for all structural Ig gene segments, RAG-1, RAG-2, and TdT, but was negative for the protein tyrosine kinase btk.
As B cells develop in the bone marrow, the pre-B cells that have recombined H chain but not L chain express membrane m chain with a "surrogate" L chain called λ5:VpreB. This membrane receptor must transduce a binding signal using btk (B cell tyrosine kinase). Without this signal, pre-B cells do not go on to recombine light chain and become mature B cells. The gene for btk is present on the X chromosome.
The pediatrician explained to Doris and Donald that their son had X-linked agammaglobulinemia (XLA). Danny would be treated with weekly injections of pooled human gamma globulin, which would help him fight infections. Boys with X-linked agammaglobulinemia usually have recurring infections with S. pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Haemophilus influenzae, pyogenic (pus-forming) bacteria. Humans normally make opsonizing antibodies to the pyogenic bacteria that promote their phagocytosis by neutrophils. People with XLA do not have problems with virus infections.
1. Flow cytometry is a technique used to quantify circulating leukocytes based on their size, granularity, and cell surface markers (see Janeway Figure A25). In the diagram at right below, forward and side scatter characteristics have been used to identify lymphocytes, monocytes and granulocytes (each dot represents one cell).
Cells labeled with fluorochrome-tagged antibodies to cell surface markers can be counted and the amount of marker on their surfaces measured.
1. Sketch the one dimensional plot for number of cells (bone marrow cells) vs. amount of membrane IgM in a normal individual. What antibody would you use to obtain this data? Now sketch the one dimensional plot for Danny's bone marrow.
2. Sketch the one dimensional plot for number of cells (peripheral blood lymphocytes, PBL) vs. amount of membrane IgM in a normal individual. What antibody would you use to obtain this data. Now sketch the one dimensional plot for Danny's blood. Are the plots for blood and PBHL the same or differnt? EXPLAIN.
2. Sketch the two dimensional dot plot for IgM and TCR on PBL for a normal individual; repeat for Danny's PBL.
3. Do you think btk is involved in the production of mature T cells? What information in the case is related to this question? How could you test Danny's T cell function? [Think about this one but don't spend a lot of time on it!]
4. How could Danny have IgG but no other Ig isotypes?
5. Class I MHC is on all nucleated cells; Class II MHC is on professional APC. Sketch the two dimensional flow diagrams for PBL for Class I vs Class II MHC; Class I MHC vs. IgM; Class II MHC vs. IgM; Class I MHC vs. TCR (CD3); Class II MHC vs. TCR.