A critical role for B cells in the development of memory CD4 cells.

Activated B cells express high levels of class II MHC and costimulatory molecules and are nearly as effective as dendritic cells in their APC ability. Yet, their importance as APC in vivo is controversial and their role, if any, in the development of CD4 memory is unknown. We compared responses of CD4 cells from normal and B cell-deficient mice to keyhole limpet hemocyanin over 6 mo and observed diminished IL-2 production by cells primed in the absence of B cells. This was due to lower frequencies of Ag-responsive cells and not to decreased levels of IL-2 secretion per cell. The absence of B cells did not affect the survival of memory CD4 cells since frequencies remained stable. Despite normal dendritic cell function, multiple immunizations of B cell-deficient mice did not restore frequencies of memory cells. However, the transfer of B cells restored memory cell development. Ag presentation was not essential since B cells activated in vitro with irrelevant Ag also restored frequencies of memory cells. The results provide unequivocal evidence that B cells play a critical role in regulating clonal expansion of CD4 cells and, as such, are requisite for the optimal priming of memory in the CD4 population.

Regulation of development and function of memory CD4 subsets.

Immunologic memory refers to the dramatic response to previously encountered antigen (Ag) that is largely controlled by CD4 T cells. Understanding how CD4 memory is regulated is essential for exploiting the immune system to protect against disease and to dampen immunopathology in allergic responses and autoimmunity. Using defined adoptive-transfer models, we are studying parameters that affect differentiation of memory CD4 cells in vivo and have found that a complex interplay of T cell receptor signaling, costimulation, and cytokines can determine the extent of memory development and the balance of Th1 and Th2 memory subsets. On challenge, memory CD4 cells localize in sites of Ag exposure and develop into effectors that regulate memory responses. We are investigating the roles of adhesion molecules, cytokines, and chemokines in the selective recruitment of CD4 memory subsets to address mechanisms by which memory T cells provide long-lasting immunity and, in our recent studies, to determine how memory CD4 cells contribute to the development of autoimmune diabetes.

Interleukin 2, but not other common gamma chain-binding cytokines, can reverse the defect in generation of CD4 effector T cells from naive T cells of aged mice.

Development of effectors from naive CD4 cells occurs in two stages. The early stage involves activation and limited proliferation in response to T cell receptor (TCR) stimulation by antigen and costimulatory antigen presenting cells, whereas the later stage involves proliferation and differentiation in response to growth factors. Using a TCR-transgenic (Tg(+)) model, we have examined the effect of aging on effector generation and studied the ability of gamma(c) signaling cytokines to reverse this effect. Our results indicate that responding naive CD4 cells from aged mice, compared with cells from young mice, make less interleukin (IL)-2, expand poorly between days 3 to 5, and give rise to fewer effectors with a less activated phenotype and reduced ability to produce cytokines. When exogenous IL-2 or other gamma(c) signaling cytokines are added during effector generation, the Tg(+) cells from both young and aged mice proliferate vigorously. However, IL-4, IL-7, and IL-15 all fail to restore efficient effector production. Only effectors from aged mice generated in the presence of IL-2 are able to produce IL-2 in amounts equivalent to those produced by effectors generated from young mice, suggesting that the effect of aging on IL-2 production is reversible only in the presence of exogenous IL-2.

Age-related changes in CD4 T cells of T cell receptor transgenic mice.

Much of the decrease in immunoresponsiveness seen in elderly populations is associated with changes in T cells responses. The observed functional changes include decreased responsiveness to T cell receptor stimulation and altered profiles of cytokine secretion. At the same time there is a decrease in the proportion of T cells that express a naive phenotype (CD44lo, CD45RBhi, CD62Lhi) and an increase in those that express a memory phenotype (CD44hi, CD45RBlo, CD62Llo). These changes are thought to result in the increased susceptibility to infection and decreased efficacy of vaccination that are observed in the elderly population. In this paper, we compare our published findings of the changes in antigen-specific T cell responsiveness using aged T cell receptor transgenic (TCR Tg) mice to what is known using conventional murine models. The specific antigen recognized by this transgenic T cell receptor apparently does not appear in the environment and the T cells expressing the Tg retain a naive phenotype. Similar to the findings in aged humans and rodents, the TCR Tg+ and TG- CD4 T cells from aged transgenic mice display decreased capacities for proliferation and cytokine secretion. Although the proportion of CD4 T cells that possess a memory phenotype increased in aged TCR Tg mice, they are Tg-. These findings support the presence of age-related deficiencies which do not depend on response to antigen. The implications of these findings are discussed.

From naive to effector--alterations with aging.

We have used a T-cell receptor transgenic mouse model to study the role of antigen in the changes that occur as T cells age. We find that the characteristic shift in the CD4 population to a predominance of memory phenotype T cells which accompanies aging in non-transgenic mice does not occur, suggesting that this shift is a result of antigenic stimulation. Thus at least one component of aging must be antigen dependent. When responses of naive CD4 T cells from aged and young mice are directly compared in vitro, the former are relatively deficient in their ability to produce IL-2 and IL-3, they express altered levels of P-glycoprotein and they proliferate less well in the absence of exogenous cytokines. When the ability of both naive populations to generate effectors is compared, the number of effectors generated from aged naive cells is much reduced and the effectors generated express lower levels of IL-2R alpha and produce reduced levels of cytokines. Importantly, addition of IL-2 restores proliferation of aged naive T cells, restores efficient effector generation and results in effectors seemingly indistinguishable from those derived from young CD4 cells. Similar phenotypic and functional changes seen with aging are also found in T-cell populations from IL-2 and IL-2R alpha knockout mice. Thus the loss of optimal IL-2 production may participate in the aging process and may represent the main antigen-independent defect in the CD4 T-cell population.

Antigen-independent changes in naive CD4 T cells with aging.

In the elderly, a dramatic shift within the CD4+ T cell population occurs, with an increased proportion having a memory phenotype with markedly decreased responsiveness. To determine what aspects of the aged phenotype are dependent upon repeated contact with antigen in the environment, we examined CD4+ cells isolated from TCR Tg mice. There is good evidence that no cross-reacting antigens for the Tg TCR recognizing pigeon cytochrome c are found in the environment of the animal, so that alterations in the Tg CD4+ cells with aging are likely to be due to antigen-independent processes. We found that in aged animals, TCR transgene(pos) CD4+ cells, although decreased in number and antigen responsiveness, maintain a naive phenotype rather than acquiring a prototypical aged memory phenotype. In contrast, the population of transgene(1o-neg) CD4+ cells increase in proportion and express the aged phenotype. Consistent with their naive status, transgene(pos) cells of aged individuals remain CD44lo CD45RBhi, secrete IL-2 and not IL-4 or IFN-gamma upon antigenic stimulation, and require co-stimulation to proliferate to anti-CD3 stimulation. These findings suggest that the aging-associated shift to CD4 cells expressing the memory phenotype is dependent on antigenic stimulation. However, the decrease in antigen responsiveness of naive transgenepos cells, as revealed by a lower secretion of IL-2 and IL-3 and a lower proliferative capacity, suggests that additional intrinsic changes occur with aging that do not depend on encounter with antigen.

Defining subsets of naive and memory B cells based on the ability of their progeny to somatically mutate in vitro.

The increased affinity of memory antibody responses is due largely to the generation and selection of memory B cells that accumulate somatic mutations after initial antigenic stimulation. Further affinity maturation and mutation also accompany subsequent immunizations. Previous studies have suggested that, like primary antibody-forming cell (AFC) clones, secondary AFC do not accumulate further mutations and, therefore, the origins of progressive affinity maturation remain controversial. Here, we report the generation of somatically mutated memory B cell clones in vitro. Our findings confirm the existence of a naive B cell subset whose progeny, rather than generating AFC, somatically mutate and respond to subsequent antigenic stimulation. Interestingly, upon stimulation, a subset of memory B cells also generates antigen-responsive cells that accumulate further somatic mutations.

Specific immunoglobulin cDNA clones produced from hybridoma cell lines and murine spleen fragment cultures by 3SR amplification.

The isothermal 3SR amplification method has been employed to assist in cloning the VL and VH genes from cells of hybridomas and splenic fragment cultures expressing antibodies for phosphorylcholine (PC) and estradiol (E2), respectively. As a first step, pools of degenerate primer pairs were identified complementary to immunoglobulin light and heavy chain variable (V) genes and capable of amplifying immunoglobulin RNA specifically at 42 degrees C. To evaluate the functionality of the 3SR-cloned immunoglobulin genes, anti-PC VH and VL cDNAs were joined together to form a single chain (sc) antibody construct and were expressed in Escherichia coli under the regulation of the alkaline phosphatase (phoA) promoter. Similarly, the combination of a murine spleen fragment and 3SR methodologies were employed to clone a selected pool of cDNAs for cultures producing anti-estradiol antibodies. This approach of using the murine spleen fragment and 3SR isothermal amplification offers the advantages of B-cell follicle architecture for antigen-driven B-cell maturation and proliferation and RNA-specific amplification, respectively. The potential utility of these advantages for the production of monoclonal antibodies and for providing the capability of studying memory B-cell development are discussed.

Among naive precursor cell subpopulations only progenitors of memory B cells originate germinal centers.

Immunization leads to the generation of both antibody-forming cells (AFC) and memory B cells which are thought to arise in germinal centers within lymphoid follicles. The findings that the precursors to memory B cells reside in the J11Dlo subpopulation of the spleens in non-immune mice and that this subpopulation is distinct from conventional AFC precursors, including CD5+ B cells, suggest that the precursors of germinal centers might also reside in the J11Dlo subpopulation. To test this hypothesis, SCID mice were repopulated with CD4+ carrier-primed T cells and T-depleted J11Dlo, J11Dhi or CD5+ B cells and immunized with a hapten-carrier conjugate. Only the J11Dlo population was enriched for cells that produced germinal centers. Thus, the subpopulation of precursors that generates memory B cells also originates germinal centers.

The generation of memory B cells.

Immunization leads to the generation of antibody forming cells (AFC) and secondary B cells which differ substantially from primary B cells. Based on the function of the progeny of enriched precursor cell populations, naive progenitors of memory B cells have been separated from primary AFC precursors. Precursors of memory cells: (1) require multiple antigenic stimulations to generate antibody responses which are prolonged, of increased magnitude, and generated with rapid kinetics; (2) have the capacity to form germinal centers; (3) accumulate somatic mutations; (4) display repertoire similarities with secondary B cells; and (5) can be stimulated with cross-reactive antigens. The primary AFC precursors responded with characteristic primary responses.

Tolerance susceptibility of newly generating memory B cells.

Newly generating memory B cells rapidly accumulate somatic mutations that can alter their Ag-combining sites and potentially engender recognition of self determinants. To investigate the possibility that, during their emergence secondary B cells pass through a window of tolerance susceptibility, we have examined the in vitro generation of memory B cells in the presence or absence of tolerogen. The findings indicate that, before antigenic stimulation, precursors to memory B cells are resistant to tolerance induction. However, 2 to 7 days after T cell-dependent antigenic stimulation, newly emerging hapten-specific secondary B cells can be inactivated by the presence of hapten on a carrier not recognized by available Th cells. This inactivation can be blocked by the presence of free hapten and can be competed by the presence of immunogen. Inactivation of newly generating secondary B cells appears less specific than the tolerance induction of immature neonatal or bone marrow B cells because inactivation can be accomplished by cross-reactive determinants. Interestingly, the presence of tolerogen after primary stimulation did not preclude the generation of cells responsive to a third in vitro stimulation. Therefore, whereas newly emerging memory B cells are highly susceptible to inactivation, the progression of the clones of progenitors to memory B cells appears resistant to tolerance induction.

Expression of mouse IgA by transgenic mice, pigs and sheep.

The development of transgenic animal technology allows the introduction of desired traits into the germ line of mice and other animals. Since the production of antibody to various polysaccharide antigens can be protective against pathogenic bacteria, we generated transgenic mice, sheep and pigs carrying genes encoding the mouse alpha and kappa chains for antibodies against phosphorylcholine (PC) to determine whether transgene antibody might be used to influence susceptibility to disease. High serum levels of mouse IgA were detected in transgenic mice and pigs but not in transgenic sheep. It has been noted that transgene immunoglobulin expression can suppress endogenous immunoglobulin gene rearrangement and expression, and indeed, in one mouse line, expression of the transgene resulted in less than 10% of spleen B cells expressing endogenous IgM. Despite this suppression, significant levels of endogenous IgM were still secreted into the serum. Suppression of endogenous IgM expression was not seen in other mouse lines, nor was it seen in transgenic pigs. In the transgenic pigs, the mouse IgA was detected in the serum despite the absence of an intact mouse kappa transgene, so the secreted antibody presumably included pig light chains. Little if any of the mouse IgA in these sera showed binding specificity for PC. In one of the founder sheep, mouse IgA was detectable in peripheral lymphocytes but not in serum. Mouse kappa expression was not detected in the transgenic sheep harboring an intact kappa transgene. These results illustrate the potential of introducing beneficial traits such as germ-line-encoded immunity into large mammalian species.

Elevated PC responsive B cells and anti-PC antibody production in transgenic mice harboring anti-PC immunoglobulin genes.

The rearrangement of heavy and light chain immunoglobulin genes is necessary for the production of functional antibody molecules. The myeloma MOPC 167 produces specific antibodies to the antigen phosphorylcholine (PC), which is present on bacterial surfaces, fungi and other environmental contaminants. Rearranged heavy and light chain immunoglobulin genes cloned from MOPC 167 were microinjected into mouse eggs. Within the resulting transgenic mice, expression of the transgenes were limited to lymphoid tissues. Transgenic mice produced elevated levels of anti-PC antibodies constitutively, at 16 days of age, when normal non-transgenic mice were not fully immunocompetent. A triggering antigenic stimulus was not necessary to evoke anti-PC immunoglobulin production. Additionally, the frequency of PC-responsive B cells in these transgenic mice was further increased upon specific immunization.

Altered VH gene segment utilization in the response to phosphorylcholine by aged mice.

Newly generated bone marrow B cell precursors of aged BALB/c mice, stimulated in splenic fragment cultures, display a markedly increased frequency of phosphorylcholine (PC)-responsive cells. This increased frequency is found for both precursors that utilize VHS107, a phenotype common to essentially all PC-specific B cells of young mice, and, surprisingly, for precursors that utilize VH genes other than VHS107. PC-specific hybridomas derived from bone marrow cells of aged mice utilize members of at least three VH gene segment families that have never been observed in PC responses of young mice. The ability of aged but not young mice to generate these unique PC-specific clonotypes may be evidence for constraints on V region utilization during repertoire development in young adults and has important implications for aging-associated changes in immune responsiveness.