Dominant Clonotypes Research Articles

Differences in the requirement of antigen‐presenting cells for long‐term growth among cytomegalovirus‐specific Th clonotypes

Functional and molecular studies of in vivo-activated T lymphocytes involved in normal and abnormal immune responses, i.e., cells infiltrating tissues affected by autoimmune processes, require their previous in vitro expansion. Problems such as unavailability of specific antigen(s) (Ag) and/or the requirement of large amounts of autologous peripheral blood mononuclear cells (PBMC) as feeders have prompted the development of alternative expansion methods that circumvent the use of antigen-presenting cells (APC) and/or Ag. We have previously shown that cytomegalovirus (CMV)-specific T cell lines and clones can be efficiently propagated in long-term cultures by stimulation with agonistic monoclonal antibodies (mAb) coated onto polystyrene particles in the absence of APC. However, this and other stimulation protocols may skew the repertoire of clonotypes that proliferate in response to nominal Ag and APC. Here we show that polyclonal populations of CMV-primed and interleukin-2 (IL-2)-stimulated PBMC undergo the same clonotypic selection when induced to grow both by continuous stimulation with CMV and an anti-CD3 mAb presented by APC. This selection, reproduced in three independent expansion experiments, involved the dominant growth of two CMV-specific, IL-2-secreting CD4+ clonotypes sharing J alpha, J beta, V alpha and V beta genes and highly homologous T cell receptor (TcR) junctional sequences. The dominant growth of these 2 clonotypes required a direct T cell/APC interaction since when coated onto polystyrene particles the same mAb induced the selective expansion of another IL-2-secreting CD4+ CMV-specific clonotype expressing a different, yet homologous, TcR heterodimer (used same V alpha and V beta genes), which was underrepresented before expansion (5 vs. 58%). T cell clones belonging to the subdominant clonotype proliferated significantly faster in response to stimulation with anti-CD3 mAb coated onto beads than in response to stimulation with CMV or anti-CD3 mAb presented by APC, possibly due to long-term expansion without APC or antigen. In contrast, neither the dominant clonotypes nor unprimed T cells were able to undergo CD3-mediated expansion in the absence of APC. We conclude that quantitative differences in growth competency among normal Ag-activated T-helper (Th) clonotypes in response to antigenic stimulation can be reproduced by stimulation through the TcR in the absence of TcR occupancy but only in the presence of APC and that certain clonotypes do not require APC for long-term growth in vitro. These data have practical implications for the isolation and repertoire characterization of in vivo-activated T cells from tissues affected by inflammatory, i.e. autoimmune, phenomena.

Human antibodies to group A streptococcal carbohydrate. Ontogeny, subclass restriction, and clonal diversity.

Abstract To investigate immuno-incompetence to polysaccharide Ag in young children, antibodies to the polysaccharide and protein Ag of Streptococcus pyogenes were studied. S. pyogenes was chosen because it commonly causes natural infections and has well-characterized polysaccharide and protein Ag. In children over the age of 2 yr it was found that the maturation of antibody responses to the polysaccharide Ag of S. pyogenes (A-CHO) appeared to occur in parallel with, or even earlier, than the responses to streptococcal protein Ag. When antibodies to group A carbohydrate (A-CHO) were studied in detail, qualitative differences between the antibodies of children and adults were demonstrated. Although anti-A-CHO antibodies of adults were strikingly restricted to the IgG2 subclass, those of children were found in both the IgG1 and IgG2 subclasses. In addition, the clonal diversity of IgG antibodies to A-CHO increased with age, and additional clonotypes were detectable in convalescent sera of some subjects of all ages after infection. Two cases with major additional clonotypes after group A streptococcal infection were studied in detail. In these two cases the additional clonotypes belonged to a different IgG subclass than the previously dominant clonotypes, and the expression of the additional major clonotypes occurred in both IgG1 and IgG2 subclasses.

Clonal dominance of low-affinity antibodies in rabbit hyperimmune anti-streptococcal group A-variant polysaccharide antisera.

Intraveneous hyperimmunization of selectively bred rabbits with streptococcal group A-variant vaccines elicits antibody responses of restricted heterogeneity at high antibody levels. In these antisera, IgG with dissociation constants Kd = 10(-6) M constitutes 90% and IgG with Kp = 10(-9) M accounts for only 10% of the group A-variant polysaccharide-specific antibodies. The low affinity antibody fraction represents the dominant clonotypes. Preparative isoelectric focusing in granulated (Ultrodex G-75) gels was used to successfully purify single-band material belonging to dominant spectrotypes. Affinity studies with these antibody fractions with the highest reported degree of purity yielded Kd = 10(-6) M values, thus confirming that clonal dominance is exclusively associated with low-affinity antibodies. Since it is known from previous work (M. Cramer and D. G. Braun, Scand. J. Immunol. 1975. 4:63) and from the rabbit antisera used here that clonal dominance of this sort is long-lived, this work fails to support the argument of immune maturation. The data more logically relate to antibodies that emerge with different subspecificities -- recognized in the antigen as a function of time in immunization procedures -- rather than to an inherent property of the immune system.