Impaired Lymphocyte Function in Aged Humans

The response of lymphocytes from young and old persons to phytohemagglutinin, pokeweed mitogen, or allogeneic lymphocytes has been measured. Lymphocytes from old persons incorporated significantly less tritiated thymidine as compared with lymphocytes from young persons when cultured with plant mitogens or allogeneic cells. The difference in observed lymphocyte reactivity could not be attributed to differences in culture conditions required for maximal transformation of lymphocytes from old or young subjects. The same percentage of thymus-derived and bone marrow-derived lymphocytes was found in the blood from old and young persons. The relationship of these findings to the decline of immunologic competence with age is discussed.     

Decreased sensitivity of old and progeric human fibroblasts to a preparation of factors with insulinlike activity.

To determine whether old cells have a reduced response to a preparation of factors from human plasma with insulinlike activity (ILA), we analyzed the response to ILA of early and late passage human fibroblasts from young, old, and progeric donors in the acute stimulation of [3H]2-deoxy-D-glucose (2dG) uptake and the delayed stimulation of [3H]thymidine (TdR) incorporation into DNA. The ILA concentration required to produce equivalent, relative stimulation of TdR incorporation was increased two- to three-fold in late passage cells and cells from old and progeric donors (P less than 0.01). 50 and 95% of maximal stimulation (ILA50, ILA95) was achieved by 0.26 +/- 0.07 and 1.38 +/- 0.13 ng insulin equivalents/ml (mean +/- SD) respectively, in cells from young adults at early passage. Corresponding values were 0.54 +/- 0.05 and 2.90 +/- 0.25 in cells from old donors; greater than 0.9 +/- 0.1 and greater than 3.1 +/- 0.1 in cells from a 9-yr-old progeric donor; and 0.4 +/- 0.05 and 1.1 +/- 0.04 in cells from normal children (9-13 yr). For two cell strains from young adults, ILA50 and ILA95 were 0.30 +/- 0.02 and 1.0 +/- 0.3 ng eq/ml at 30% of their in vitro lifespan completed (%LC) and these values increased at rates of 0.005 ng eq/ml per %LC and 0.04 ng eq/ml per %LC, respectively. The mean stimulation of 2dG uptake ratio (ILA/control) decreased from early to late passage from 2.1 +/- 0.6 to 1.3 +/- 0.1 in young adult donors (P less than 0.05), but there were no significant differences between young and old donors at either early or late passage. The mean stimulation ratio in progeric cells (1.2 +/- 0.2) did not change with in vitro passage, but was significantly lower than that of age-matched normal cells (2.1 +/- 0.8, P less than 0.001). In progeria cells, the reduced stimulation of 2dG uptake upon addition of ILA was due to an increased basal rate of uptake (0.19 +/- 0.01 pmol [3H]2dG/min per mg protein vs. 0.13 +/- 0.01 in age-matched normal cells), and not to a decline in the maximal rate of uptake (0.26 +/- 0.01 vs. 0.27 +/- 0.02, respectively). Similar results were found for in vitro aging in cells from an old donor.     

Erythropoiesis in the aged mouse: II. Response to stimulation in vitro

In this study, we present in vitro evidence that erythropoietic precursors in aged mice respond less to stimulation by erythropoietin than do precursors in young mice. The effect of age on proliferation of differentiated erythroid cells from the marrow of young and old mice was examined in liquid culture to which increasing concentrations of erythropoietin were added. Cellular proliferation was measured indirectly by 59Fe incorporation into heme and directly as tritiated thymidine incorporation into DNA. The number of normoblasts remaining in culture with and without the addition of erythropoietin was also measured. In each case, cellular proliferation was significantly lower in marrow in old than in young mice. In contrast, CFU-E colonies cultured with increasing doses of erythropoietin were similar in young and old animals. These findings indicate that aging causes a reduction in the proliferative response of differentiated erythroid cells. Failure of these cells to respond to stimulation is the likely mechanism for the reduced erythropoietic proliferative capacity found in aged animals.     

Production of auto-antiidiotypic antibody during the normal immune response. VII. Analysis of the cellular basis for the increased auto- antiidiotype antibody production by aged mice

We have previously shown that old mice produce more hapten-augmentable plaque-forming cells (PFC) than do young animals, suggesting a greater auto-antiidiotype antibody (auto anti-Id) component in their immune response. In the present studies this is confirmed serologically. The marked auto-anti-Id response of aged mice can be transferred to lethally irradiated young recipients with spleen but not bone marrow cells from old donors, suggesting that it is an intrinsic property of their peripheral B cell population and that the distribution of Id arising from the bone marrow of old and young mice is similar. In contrast with young mice the auto-anti-Id response of old animals is relatively T cell-independent and old donors do not show an increase in their ability to transfer an auto-anti-Id response after priming with TNP-F. These observations suggest that old mice behave as if already primed for auto-anti-Id production. Irradiated mice reconstituted with bone marrow cells from either young or old donors together with splenic T cells from old donors generate a relatively large auto-anti-Id response, whereas mice reconstituted with bone marrow from either young or old donors together with splenic T cells from young donors produce few hapten-augmentable PFC. It is suggested that differences in Id expression and auto-anti-Id production are the consequences of the interaction of Id (and anti-Id) arising from the marrow with anti-Id (and Id) present in the peripheral T cell population which serves as a repository of information about shifts in Id distribution, resulting from lifelong interactions with environmental and self-antigens.     

Immunological studies of Aging. III. Cytokinetic basis for the impaired response of lymphocytes from aged humans to plant lectins

The basis for the age-associated defect in the response of lymphocytes to plant lectins has been studied. Using three independent assays we have shown that the number of mitogen-responsive cells is markedly reduced in lymphocyte preparations from old persons. In addition, studies using colchicine bloock and thymidine pulse techniques have revealed a failure of mitogen-responsive cells from old persons to expand into a proliferating pool of lymphocytes as is observed when lymphocytes from young persons are cultured with phytohemagglutinin. Thus, the impaired response of lymphocytes from old persons to mitogens is attributable to a reduced number of mitogen responsive cells and their failure to undergo clonal expansion.     

Responsiveness of peripheral blood lymphocytes from cancer patients and healthy donors to interleukin-2 (IL-2)

The proliferative response of the peripheral mononuclear cells (MNC) from cancer patients and healthy individuals to IL-2 was studied by use of the quantitative microwell assay of the 3H-TdR incorporation into the cells in vitro. After the addition of phytohemagglutinin (PHA), MNC acquired the reactivity to IL-2 within 3 hr, and reached a maximum after 12 to 17 hr of incubation. Although the IL-2 response of PHA-activated MNC from cancer patient was lower than that from healthy donor, there was no significant difference in the kinetics of proliferation. The maximum response of PHA-activated MNC from both cancer patients and healthy donors to IL-2 was observed at 96 hr and 84 hr, respectively. When monocytes were removed from MNC, IL-2-associated growth of the remaining fraction of MNC was decreased to 40-70% in both cancer patients and healthy donors. Furthermore, monocytes from cancer patients did not affect the IL-2 responsiveness of lymphocytes from healthy donors, and vice versa. The number of T lymphocytes having IL-2 receptors (IL-2R) from cancer patients was lower than that from healthy donors. These facts indicated that the lower IL-2 response of MNC from cancer bearer was due to the decreased number of T lymphocytes possessing IL-2R, and not due to monocytes themselves.     

Phytohemagglutinin Response in Systemic Lupus Erythematosus: RECONSTITUTION EXPERIMENTS USING HIGHLY PURIFIED LYMPHOCYTE SUBPOPULATIONS AND MONOCYTES

THE PHYTOHEMAGGLUTININ (PHA) RESPONSE OF LYMPHOCYTES FROM UNTREATED PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) WAS STUDIED USING HIGHLY PURIFIED SUBPOPULATIONS OF CELLS INVOLVED IN THE TRANSFORMATION RESPONSE: T lymphocytes, B lymphocytes, and monocytes. Cell transformation was quantitated using both tritiated thymidine ([(3)H]-TdR) incorporation into DNA and cytofluorographic determination of cellular DNA content. Dose-response curves using six concentrations of PHA and five concentrations of cells over 0-5 days revealed a decrease in [(3)H]TdR by stimulated lymphocytes from some SLE patients. This decrease in [(3)H]TdR was paralleled by a decreased percentage of cells in S, G(2), and M phases of the cell cycle. However, abnormal response occurred entirely in those SLE patients who were hypocomplementemic. The etiology of the impaired response was further examined. Lymphocyte receptors for concanavalin A were studied using cytofluorography of lymphocytes stained with fluorescein-conjugated concanavalin A. The frequency distribution of concanavalin A receptors was similar in the normocomplementemic and hypocomplementemic lupus patients and in normals. The latex phagocytic activity of lupus macrophages was similar to normals when allogeneic normal plasma was used in the culture medium. Phagocytic activity became abnormal in the presence of SLE plasma. However, there was no difference in the [(3)H]TdR response or the percentage of cells in S, G(2), and M phases when T lymphocytes from the hypocomplementemic patients were stimulated on either autologous or normal allogeneic monocyte monolayers. Likewise, normal lymphocytes incorporated similar amounts of [(3)H]TdR and had similar percentages of cells in S, G(2), and M phases whether their T lymphocytes were stimulated on autologous or SLE monocyte monolayers. Highly purified subpopulations of B and T lymphocytes were obtained by density sedimentation or Fenwal Leuko-Pak passage of lymphocyte populations. The response to PHA by lymphocytes from the hypocomplementemic lupus patients could be seen to involve at least two abnormalities. One, in reference to normal lymphocytes, SLE T lymphocytes plus monocytes had an impaired response; two, SLE B lymphocytes plus SLE T lymphocytes plus SLE monocytes had an impaired response. Two patients in the hypocomplementemic group were treated with steroids. 5 days after steroid treatment was initiated, the percentage of cells in S, G(2), and M phases and the [(3)H]TdR response of PHA-stimulated lymphocytes returned to normal. The normalization of the [(3)H]TdR response was explained both by a return of purified T cells plus monocytes, purified B cells plus monocytes, and whole lymphocyte populations to normal responsiveness. These studies suggest that a steroid-correctable defect exists in T and B lymphocytes in SLE.     

Studies on a new lymphocyte mitogen from Bordetella pertussis. I. Induction of proliferation and polyclonal antibody formation

Pertussis B mitogen (PBM), isolated from culture supernatant fluids of Bordetella pertussis, is a potent mitogen for mouse and human lymphocytes. In mice, > 95% of the blast cells recovered from PBM cultures bear surface immunoglobulins. Therefore, PBM seems to induce proliferation of mouse B lymphocytes, but not T cells. The proliferative response observed is nonspecific because cells from all mouse strains tested, including germfree animals, are responsive. Moreover, the mitogenic activity of PBM is independent of T lymphocytes, macrophages, or serum factors. When human peripheral blood or cord blood lymphocytes are cultured in the presence of PBM, a high level of thymidine incorporation by these cells is detected. Furthermore, PBM can induce polyclonal antibody formation by both mouse and human lymphocytes. Despite similar methods of isolation, PBM is distinct from the lymphocytosis-promoting factor of B. pertussis, a previously described T cell mitogen.     

CELLULAR INTERACTIONS IN THE PROLIFERATIVE RESPONSE OF HUMAN T AND B LYMPHOCYTES TO PHYTOMITOGENS AND ALLOGENEIC LYMPHOCYTES

In vitro studies were performed to determine the proliferative responsiveness of human peripheral blood thymus-dependent (T) and thymus-independent (B) lymphocytes to phytomitogens and allogeneic lymphocytes. Recombination of T and B cells, with selective inhibition of proliferation of one of the two populations, was used to identify cellular interactions which may contribute to cell proliferation. The distinctive feature of human T lymphocytes to form rosettes with unsensitized sheep erythrocytes was utilized to separate human peripheral blood lymphocytes into highly purified resetting (T) and non-rosetting (B) cells. The proliferative response of these separated lymphocyte subpopulations to various stimulants was assessed from the uptake of tritiated thymidine into DNA. Phytohemagglutinin, concanavalin A, pokeweed mitogen, and allogeneic lymphocytes stimulated separated T cells, whereas no proliferation was observed with the T-cell-depleted B-cell population. This suggests that it is the human T cell which is activated directly by these stimulants. In the presence of T cells (proliferating or nonproliferating), B cells were capable of proliferation following stimulation with phytomitogens, but not in response to histocompatibility antigens. Thus, T-cell-mediated B-cell proliferation contributes to the overall lymphocyte response in phytomitogen-stimulated T + B cell mixtures, but not in human mixed leukocyte cultures. T-cell activation by allogeneic cells required the presence of monocytes; in contrast, the three tested phytomitogens stimulated T cells in the absence of monocytes. This indicates that direct interaction of mitogens with lymphocyte membrane receptors is sufficient to trigger T cells into proliferative response. However, monocytes considerably enhanced the proliferative response of T cells in a dose-dependent fashion; this monocyte-dependent mechanism of T-cell activation was predominant at lower concentrations of phytomitogens, and contributed relatively less at higher mitogen doses. Both, the direct, monocyte-independent, and the indirect, monocyte-dependent T-lymphocyte activation contribute to the total in vitro response of lymphocyte preparations to phytomitogens.     

Stimulation of human dermal fibroblasts with interleukin 2

The role of soluble inflammatory mediators in the wound healing process is under investigation. Interleukin 2 (IL2), an immune modulator produced by T lymphocytes, was tested in vitro for its effects on human diploid fibroblasts. Human diploid fibroblasts (1.0 x 10(4)) were incubated for 24 hours in Alpha minimal essential media (Gibco Laboratories, Gaithersburg, Md.) at 37 degrees C in 5% CO2 in air with 500 U/ml of IL2 (Cetus Corp., Emeryville, Calif.). Cells were then pulsed with 3 mu Ci/ml of tritiated thymidine overnight. The mean increase in the incorporation of tritiated thymidine of the cells treated with IL2 over control cells was 38% (p value less than 0.05). Interleukin 2 has a significant effect on the metabolism of human dermal fibroblasts and may accelerate the wound healing process.     

T cell growth factor receptors. Quantitation, specificity, and biological relevance

To examine directly the hypothesis that T cell growth factor (TCGF) interacts with target cells in a fashion similar to polypeptide hormones, the binding of radiolabeled TCGF to various cell populations was investigated. The results indicate that TCGF interacts with activated T cells via a receptor through which it initiates the T cell proliferative response. Internally radiolabeled TCGF, prepared from a human T leukemia cell line and purified by gel filtration and isoelectric focusing, retained biological activity and was uniform with respect to size and charge. Binding of radiolabeled TCGF to TCGF-dependent cytolytic T cells occurred rapidly (within 15 rain at 37 degrees C) and was both saturable and largely reversible. In addition, at 37 degrees C, a receptor- and lysosome-dependent degradation of TCGF occurred. Radiolabeled TCGF binding was specific for activated, TCGF-responsive T cells. Whereas unstimulated lymphocytes of human or murine origin and lipopolysaccharide-activated B cell blasts expressed few if any detectable binding sites, lectin- or alloantigen-activated cells had easily detectable binding sites. Moreover, compared with lectin- or alloantigen-activated T cells, long-term TCGF-dependent cytolytic and helper T cell lines and TCGF-dependent neo-plastic T cell lines bound TCGF with a similar affinity (dissociation constant of 5-25 pM) and expressed a similar number of receptor sites per cell (5,000-15,000). In contrast, a number of TCGF-independent cell lines of T cell, B cell, or myeloid origin did not bind detectable quantities of radiolabeled TCGF. Binding of radiolabeled TCGF to TCGF-responsive cells was specific, in that among several growth factors and polypeptide hormones tested, only TCGF competed for binding. Finally, the relative magnitude of T cell proliferation induced by a given concentration of TCGF closely paralleled the fraction of occupied receptor sites. As the extent of T cell clonal expansion depends on TCGF and on the TCGF receptor, the dissection of the molecular events surrounding the interaction of TCGF and its receptor that these studies permit, should provide new insight into the hormonelike regulation of the immune response by this lymphokine.     

Requirement of precommitted cells as targets for the augmentation of lymphocyte proliferation by leukocyte dialysates

After our initial report tha leukocyte dialysates containing transfer factor augment the thymidine incorporation of antigen-stimulated lymphocytes, we have adapted the system to microleukocyte cultures. This modification permits both (a) the simultaneous assay of a single dialysate on the cells of multiple individuals, and (b) the assay of multiple dialysates on the cells of a single individual. The data thus secured, demonstrate that dialysates from both skin-test-positive and -negative donors produced similar degrees of augmentation whether the data are expressed as an arithmetic difference or as a ratio. When expressed as an arithmetic difference, the amount of augmentation is increased in proportion to the level of thymidine incorporation of the assay cells when they were stimulated by antigen alone. When expressed as a ratio, however, the degree of augmentation is independent of the response of the assay cells. An analysis of the ability of dialysates to engage previously uncommitted lymphocytes and thus to augment thymidine incorporation, revealed that precommitted cells were required. In these experiments, antigen-reactive cells were deleted from populations of peripheral blood lymphocytes by incubation with purified protein derivative of tuberculin, diphtheria toxoid, or streptokinase-streptodornase in the presence of [3H]thymidine of high specific activity. This deletion depressed or abolished the effect of dialysate on the residual population when it was recultured with the same antigen, but the effect on the response of the remaining lymphocytes to other antigens was unaltered. In this study, leukocyte dialysate appeared to augment nonspecifically the thymidine incorporation of an antigen-specific precommitted clone of lymphocytes. The relationship of these adjuvant effects on peripheral blood lymphocytes in vitro to the specific and nonspecific activities of transfer factor in vivo remains to be elucidated.     

Suppressor cell activity after concanavalin A treatment of lymphocytes from normal donors

Pretreatment of normal human peripheral blood lymphocytes with the plant lectin, concanavalin A (Con A), results in inhibition of blast transformation and [3H]thymidine incorporation by untreated allogeneic lymphocytes from healthy volunteers donors in one-way mixed leukocyte culture. Similarly, responses to mitogens, certain microbial antigens, and allogeneic lymphocytes are inhibited by Con A-treated allogeneic cells. Con A pretreated autologous lymphocytes can also be induced to manifest suppressor activities. This antimitotic effect occurs without evidence of cytotoxicity and is active on de novo lymphocyte responses and does not require prior sensitization of the cells being tested. Suppression of the lymphocyte response to pokeweed mitogen, a potent B-cell stimulator, by Con A-pretreated suppressor cells was not as consistent as was inhibition of response to other mitogens, including phytohemagglutinin and Con A. Furthermore, suppression of lymphocyte transformation to the microbial antigens, tuberculin purified protein derivative, and Canadida albicans extracts could be similarly induced by Con A pretreatment of either allogeneic or autologous cells. Induction of autologous suppressor activity in lymphocytes from healthy donors is compatible with a model that includes a role for suppressor cells in the modulation of the normal immune response.     

Aging and antimicrobial immunity. Impaired production of mediator T cells as a basis for the decreased resistance of senescent mice to listeriosis

Immunity to infection of mice with the facultative, intracellular pathogen Listeria monocytogenes was employed as a model system to investigate the immunological basis for the age-associated decline in anti-microbial immunity. In response to a sublethal immunizing infection, aged (24-mo old or more) mice displayed a smaller increase in spleen weight, spleen cellularity, and splenic T cell content than young (3- to 4-mo-old) mice. Aged mice also generated a smaller number of anti-Listeria protective T cells at the time of a peak response, in that their spleen cells were 1,000-fold less protective than equivalent numbers of spleen cells from the young donors, even when enriched T cell populations were employed. These results suggest that the impaired ability of aged mice to produce protective T cells is mainly responsible for decreased resistance of these mice to infection with Listeria.     

Aging and arteriosclerosis. The increased proliferation of arterial smooth muscle cells isolated from old rats is associated with increased platelet-derived growth factor-like activity

In vivo studies have suggested that the aorta from an old animal responds to injury with an exaggerated proliferation of smooth muscle cells (SMCs) compared with the response of this aorta from a young animal. In this study we compared proliferation of SMCs derived from uninjured old (less than 19 mo) and young (3-4 mo) rat aortas. Old SMCs grew more rapidly than young SMCs in the presence of medium containing competence factors (10% FCS or platelet-derived growth factor [PDGF]) as well as in their absence (2% PDS or serum-free media) as determined both by a short-term thymidine incorporation assay and by cell counts. Lysates prepared from old SMCs that had been grown in the absence of serum or PDGF stimulated proliferation of target cells more than lysates prepared from young SMCs; the effect was inversely related to cell density of the SMCs. This stimulatory effect of lysates was completely blocked by antibody to PDGF. After the growth-promoting activity of lysates was eliminated by anti-PDGF, growth-inhibiting activity was revealed. Lysates prepared from old SMCs had significantly less capacity to inhibit target cell growth. In the presence of exogenous heparin both the serum- or PDGF-stimulated proliferation and serum-free proliferation of old SMCs were decreased to the level of proliferation of young SMCs. These results suggest that the balance between growth-promoting and growth-inhibiting factors is altered in SMCs from old rats. This may contribute to the increased proliferative capacity of these cells in culture and may facilitate the development of atherosclerosis with age.     

Alloantisera with apparent HLA-A specificity reacting only against phytohemagglutinin-activated cells

Summary Seven alloantisera were selected showing the following characteristics: after platelet absorption they were positive with PHA-activated cells and negative with resting T and B lymphocytes from the same donors; this reactivity could be blocked by pretreatment of cells with a turkey anti-human β₂-microglobulin serum and was not completely removed by further absorption with high doses of pooled platelets or with T or B lymphocytes from the positive donors; the reactivity of some of these antisera correlated with HLA-A antigens in a panel of PHA-activated cells and segregated with HLA haplotypes in families. It is still to be proven if the HLA determinants recognized on PHA-activated cells by these antisera are new epitopes of A,B,C molecules appearing upon activation or if they are carried by products of a new polymorphic locus linked to the HLA system. This work was supported byMinistero della Pubblica Istruzione (60%) and byRegione Piemonte, Ricerca Finalizzata № 36.     

Structural and functional characterization of the human T lymphocyte receptor for insulin-like growth factor I in vitro.

Growth factor receptors for T lymphocytes, such as interleukin 2 and insulin, are present on activated but not resting T lymphocytes. We sought to determine if insulin-like growth factor I (IGF-I) could act as a growth factor for human T cells and to characterize its receptor on resting and activated cells. Recombinant IGF-I induced two separate functions. It was chemotactic for and increased incorporation of tritiated thymidine into both unactivated (resting) and mitogen-activated T cells. High-affinity 125I-IGF-I binding to human T cells was saturable with an apparent Kd of 1.2 +/- .6 X 10(-10) M for binding to activated T cells and 1.2 +/- .9 X 10(-10) for unactivated T cells. The calculated binding for activated cells was 330 +/- 90 and for resting cells 45 +/- 9 high-affinity receptor sites per cell. Affinity cross-linking of 125I-IGF-I to resting or activated T cells revealed a radioligand-receptor complex of 360,000 mol wt when analyzed by SDS-PAGE without reduction and complexes of 270,000 and 135,000 mol wt upon reduction; prior incubation with excess unlabeled IGF-I prevented formation of the 125I-IGF-I receptor complex. Our data suggest that both resting and activated T lymphocytes bear functional IGF-I receptors similar to those found in other tissues. These receptors may mediate T cell growth and chemotaxis.     

Replication of herpes simplex virus in human B lymphocytes stimulated by Epstein-Barr virus.

Human B lymphocytes prepared from adult blood or cord blood by the use of neuraminidase-treated sheep red blood cells which did not respond mitogenically to phytohemagglutinin or pokeweed mitogen, responded well to the B95-8 strain of Epstein-Barr virus, but not to the P3HR-1 strain, with increased incorporation of tritiated thymidine. Replication of herpes simplex virus could be demonstrated in cultures of B lymphocytes preincubated with the B95-8 strain.     

Relative contribution of T and B cells to hypermutation and selection of the antibody repertoire in germinal centers of aged mice

The immune system of aged individuals often produces antibodies that have lower affinity and are less protective than antibodies from young individuals. Recent studies in mice suggested that antibodies produced by old individuals may be encoded by distinct immunoglobulin (Ig) genes and that the somatic hypermutation process in these individuals is compromised. The present study employed Ighb scid mice reconstituted with normal lymphocytes from young (2-3-mo-old) and aged (20-25-mo-old) donors and immunized with a protein conjugate of the hapten (4-hydroxy- 3-nitrophenyl)acetyl (NP) to determine whether the molecular changes in antibody repertoire reflect senescence in the B cells or whether they are mediated by the aging helper T lymphocytes. The NP-reactive B cells from splenic germinal centers (GC) were recovered by microdissection of frozen tissue sections and their rearranged Ig heavy chain variable region (VH) genes of the V186.2/V3 families were sequenced. It was found that the VH gene repertoire of the GC B cells was strongly influenced by the source of the CD4+ T cells. When T cells were donated by young mice, the anti-NP response in GC was dominated by the canonical V186.2 gene, even if the responder B cells came from aged donors. However, when the mice were reconstituted with T cells from aged donors, the expression of the V186.2 gene by young B cells was diminished and the response was dominated by the C1H4 gene, another member of the V186.2/V3 family. In contrast, the somatic hypermutation process in the GC B cells followed a different pattern. The mutation frequencies in the animals that were reconstituted with both B and T cells from young donors (1/50 to 1/150 bp) were comparable to the frequencies previously reported for NP-immunized intact young/adult mice. However, when either lymphocyte subset was donated by the aged mice, the mutation frequencies declined. Thus, mice reconstituted with T cells from the aged and B cells from the young had severely compromised mutational mechanism. Likewise, the recipients of aged B and young T cells had diminished mutations even though the repertoire of their anti-NP response was dominated by the canonical V186.2 gene. It appears that the change in germine-encoded repertoire and the decrease of somatic hypermutation represent distinct mechanisms of immunosenescence and that the aging of helper T cells plays a pivotal role in both of these processes.