restimulation, which is partially inhibited by anti- 
IL-2 and partially by anti-IL-4 antibodies. This pro- 
liferation is sustained and becomes more IL-4 de- 
pendent over time. This suggests that most immedi- 
ate-recall clonal expansion occurs when a cell is 
16 A high density Analysis of the helper T cell func- 
tion of the two subsets of CD4 T cells indicates that 
hapten-specific helper activity is predominantly in 
the I6A-I0W population. Primed l6A-low cells assist 
very well for IgM and IgGl responses and less well 
for IgG2a and IgG2b in the time frame tested. 
These findings are consistent with the known 
helper function of the cytokines IL-4, IL-5, and 
IFN-7, which are produced by I6A low-density 
CD4 T cells. 
The question of where Thl and Th2 cells fit 
within the 16A subsets still remains. Because I6A- 
low cells produce cytokines characteristic of Thl 
and Th2, the progeny of individual cells (short-term 
cloned lines) derived from the I6A population 
were tested for cytokine release upon stimulation 
with antigen. Some of the cloned lines released IL-4 
and IL-5 but not IL-2 or IFN-7; these lines are sim- 
ilar to Th2 cells, as originally defined. Some of the 
cloned lines released IFN-7 but not IL-4 and IL-5; 
these lines are similar to Thl cells. Some monoclo- 
nal T cells released all three cytokines, suggesting 
an intermediate phenotype. These data suggest that 
the I6A-I0W population is fiinher subdivided into 
Th2 and Thl cells and perhaps other phenotypes. 
In the future, Dr. Bottomly will examine how 
I6A-I0W cells mature into the equivalent of Thl 
and Th2 cells and to what extent intermediate 
forms exist in vivo. This is of great importance in 
protective immunity, as has been shown by studies 
of leishmaniasis and leprosy. 
III. CD45 Isoforms May Be Predictive of Functional 
Capabilities of a CD4 T CeU. 
Previous studies have shown that most of the 
antibodies used to subset CD4 T cells recognize 
CD45. An analysis of the specificity of binding was 
undertaken, to determine whether I6A was similar 
PUBLICATIONS 
or identical to other anti-CD45 antibodies. CD45 
molecules are quite heterogeneous in terms of 
their molecular weight, partly because of differ- 
ences in glycosylation and partly because of differ- 
ential splicing of three variable exons encoding an 
extracellular, amino portion of the molecule. By dif- 
ferential splicing, one gene can give rise to eight 
predicted isoforms. By transfecting cDNAs repre- 
sentative of several of the isoforms, it is possible to 
determine which isoform is required for antibody 
binding. In this way it was shown that the second 
variable exon is required for I6A reactivity, and 
therefore I6A does not bind to all possible CD45 
isoforms. This antibody differs from those directed 
at the human CD45 molecule, which may explain 
the differences seen between species. Because I6A 
discriminates between IL-2 and IL-4 producers in 
normal CD4 populations and between cloned lines 
of Thl and Th2, functionally distinct cells may ex- 
press characteristic isoforms of CD45. These iso- 
forms can be identified either by monoclonal anti- 
bodies that distinguish between isoforms or by 
polymerase chain reaction analysis of mRNAs en- 
coding different isoforms. By both types of analysis, 
Th2 cells express the high -molecular- weight iso- 
forms of CD45, including the three-variable exon 
form, whereas Thl cells express only the low-mo- 
lecular-weight forms, especially the no-exon form. 
This dramatic difference in the CD45 phenotype of 
these functionally distinct panels of Thl and Th2 
cells suggests that shifting of CD45 isoforms is pre- 
dictive or a result of functional differences between 
CD4 T cells. 
Future studies will focus on the developmental 
relationship between these CD4 subsets and the 
mechanism by which a proliferating cell diflferenti- 
ates into an effector cell and switches the panel of 
cytokines released. In particular this laboratory will 
focus on understanding the mechanism by which 
the immune response selects the effector mode 
that is dominant in a particular immune response. 
Dr. Bottomly is also Associate Professor of Im- 
munobiology at Yale University School of Medicine. 
Articles 
Bottomly, K. 1988. A functional dichotomy in CD4"'" T lymphocytes. Immunol Today 9:268-274. 
Bottomly, K. , Luqman, M. , Greenbaum, L., Carding, S. , West, J. , Pasqualini, T, and Murphy, D.B. 1989- A 
monoclonal antibody to murine CD45R distinguishes CD4 T cell populations that produce different 
cytokines. Eur J Immunol 19:617-623- 
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