Older age of childhood type 1 diabetes onset is associated with islet autoantibody positivity >30 years later: the Pittsburgh Epidemiology of Diabetes Complications Study.

AIMS: To examine the association between islet autoantibody positivity and clinical characteristics, residual ß-cell function (C-peptide) and prevalence of complications in a childhood-onset (age <17 years), long-duration (≥32 years) type 1 diabetes cohort. METHODS: Islet autoantibodies (glutamic acid decarboxylase, insulinoma-associated protein 2 and zinc transporter-8 antibodies) were measured in the serum of participants who attended the 2011-2013 Pittsburgh Epidemiology of Diabetes Complications study follow-up examination (n=177, mean age 51 years, diabetes duration 43 years). RESULTS: Prevalences of islet autoantibodies were: glutamic acid decarboxylase, 32%; insulinoma-associated protein 2, 22%; and zinc transporter-8, 4%. Positivity for each islet autoantibody was associated with older age at diabetes onset (glutamic acid decarboxylase antibodies, P=0.03; insulinoma-associated protein 2 antibodies, P=0.001; zinc transporter-8 antibodies, P<0.0001). Older age at onset was also associated with an increasing number of autoantibodies (P = 0.001). Glutamic acid decarboxylase antibody positivity was also associated with lower HbA1c (P = 0.02), insulinoma-associated protein 2 antibody positivity was associated with lower prevalence of severe hypoglycaemic episodes (P=0.02) and both distal and autonomic neuropathy (P=0.04 for both), and zinc transporter-8 antibody positivity was associated with higher total and LDL cholesterol (P=0.01). No association between autoantibody positivity and C-peptide was observed. CONCLUSIONS: The strong association between islet autoantibody positivity and older age at type 1 diabetes onset supports the hypothesis of a less aggressive, and thus more persistent, immune process in those with older age at onset. This observation suggests that there may be long-term persistence of heterogeneity in the underlying autoimmune process.

Predictors of early renal function decline in adults with Type 1 diabetes: the Coronary Artery Calcification in Type 1 Diabetes and the Pittsburgh Epidemiology of Diabetes Complications studies.

AIM: Diabetic kidney disease is one of the leading complications of Type 1 diabetes, but its prediction remains a challenge. We examined predictors of rapid decline in estimated GFR (eGFR) in two Type 1 diabetes cohorts: the Coronary Artery Calcification in Type 1 Diabetes (CACTI) and the Pittsburgh Epidemiology of Diabetes Complications (EDC). METHODS: A select subset of participants (CACTI: n = 210 and EDC: n = 98) diagnosed before 17 years of age with Type 1 diabetes duration ≥ 7 years, and follow-up data on eGFR by CKD-EPI creatinine for up to 8 years were included in the analyses. Early renal function decline was defined as annual decline in eGFR ≥ 3 ml/min/1.73 m2 , and normal age-related decline as eGFR ≤ 1 ml/min/1.73 m2 . Parallel logistic regression models were constructed in the two cohorts. RESULTS: Early renal function decline incidence was 36% in CACTI and 41% in EDC. In both cohorts, greater baseline eGFR (CACTI: OR 3.12, 95% CI 1.97-5.05; EDC: OR 1.92, 95% CI 1.17-3.15 per 10 ml/min/1.73 m2 ) and log albumin-to-creatinine (ACR) (CACTI: OR 3.24, 95% CI 1.80-5.83; EDC: OR 1.87, 95% CI 1.18-2.96 per 1 unit) predicted greater odds of early renal function decline in fully adjusted models. Conversely, ACE inhibition predicted lower odds of early renal function decline in women in CACTI, but similar relationships were not observed in women in EDC. CONCLUSIONS: A substantial proportion of people with Type 1 diabetes in the EDC and CACTI cohorts experienced early renal function decline over time. ACE inhibition appeared to be protective only in women in CACTI where the prevalence of its use was twofold higher compared with the EDC.

Antivibration gloves: effects on vascular and sensorineural function, an animal model.

Anti-vibration gloves have been used to block the transmission of vibration from powered hand tools to the user, and to protect users from the negative health consequences associated with exposure to vibration. However, there are conflicting reports as to the efficacy of gloves in protecting workers. The goal of this study was to use a characterized animal model of vibration-induced peripheral vascular and nerve injury to determine whether antivibration materials reduced or inhibited the effects of vibration on these physiological symptoms. Rats were exposed to 4 h of tail vibration at 125 Hz with an acceleration 49 m/s(2). The platform was either bare or covered with antivibrating glove material. Rats were tested for tactile sensitivity to applied pressure before and after vibration exposure. One day following the exposure, ventral tail arteries were assessed for sensitivity to vasodilating and vasoconstricting factors and nerves were examined histologically for early indicators of edema and inflammation. Ventral tail artery responses to an α2C-adrenoreceptor agonist were enhanced in arteries from vibration-exposed rats compared to controls, regardless of whether antivibration materials were used or not. Rats exposed to vibration were also less sensitive to pressure after exposure. These findings are consistent with experimental findings in humans suggesting that antivibration gloves may not provide protection against the adverse health consequences of vibration exposure in all conditions. Additional studies need to be done examining newer antivibration materials.

Femoral-gluteal adiposity is not associated with insulin sensitivity in type 1 diabetes.

AIMS: To quantify and compare associations between femoral-gluteal adiposity and insulin sensitivity in adults with Type 1 diabetes mellitus with adults with normal glucose tolerance. METHODS: Individuals with Type 1 diabetes (n = 28) were recruited from the Pittsburgh Epidemiology of Diabetes Complication study, a 24-year prospective study of childhood-onset diabetes, and compared cross-sectionally with individuals with normal glucose tolerance (n = 56) of similar age, sex and BMI. Insulin sensitivity was defined as whole-body glucose disposal measured by hyperinsulinaemic-euglycaemic clamps. Adiposity was quantified by dual energy X-ray absorptiometry. RESULTS: Individuals with Type 1 diabetes exhibited lower insulin sensitivity (5.8 vs. 8.2 mg min(-1) kg fat-free mass(-1), P < 0.01), lower total fat mass (20.1 vs. 29.0 kg, P < 0.001) and lower proportional leg fat mass (36.0 vs.37.7%, P = 0.03), but similar proportional trunk fat (% trunk fat mass) compared with individuals with normal glucose tolerance. Overall, results from linear regression demonstrated that higher % leg fat mass (P < 0.01) and lower % trunk fat mass (P < 0.01) were independently associated with lower insulin sensitivity after adjustments for age, sex, height, total fat mass (kg) and diabetes status. Higher % leg fat mass was independently associated with higher insulin sensitivity in individuals with normal glucose tolerance (P < 0.01) after similar adjustment; significant associations were not observed in Type 1 diabetes. CONCLUSIONS: Reduced insulin sensitivity is a prominent feature of Type 1 diabetes and is associated with total and abdominal adiposity. Compared with adults with normal glucose tolerance, leg fat mass does not show any positive association with insulin sensitivity in Type 1 diabetes.

In the absence of renal disease, 20 year mortality risk in type 1 diabetes is comparable to that of the general population: a report from the Pittsburgh Epidemiology of Diabetes Complications Study.

AIMS/HYPOTHESIS: The FinnDiane Study has reported that mortality in type 1 diabetes is not increased over a 7 year follow-up in the absence of renal disease (RD). Using the Pittsburgh Epidemiology of Diabetes Complications (EDC) Study population (n = 658) of childhood-onset type 1 diabetes (age <17 years), the present study sought to replicate and expand these findings to a 20 year follow-up (as of 1 January 2008) and examine cause of death by renal status. METHODS: At baseline (1986-1988), mean age and duration of diabetes were 28 and 19 years, respectively. RD was defined as an albumin excretion rate ≥20 µg/min from multiple samples and grouped as microalbuminuria (MA; 20-200 µg/min), overt nephropathy (ON; >200 µg/min), or end stage renal disease (ESRD; dialysis or renal transplantation). RESULTS: At baseline, 311 (47.3%) individuals had RD (MA 21.3%, ON 22.2% and ESRD 3.8%). During a median 20 year follow-up, there were 152 deaths (23.1%). Mortality was 6.2 (95% CI 5.2-7.2) times higher than expected, with standardised mortality ratios of 2.0 (1.2-2.8) for normoalbuminuria (NA); 6.4 (4.4-8.4) for MA; 12.5 (9.5-15.4) for ON; and 29.8 (16.8-42.9) for ESRD. Excluding those (n = 64) with NA who later progressed to RD, no significant excess mortality was observed in the remaining NA group (1.2, 0.5-1.9), whose deaths were largely unrelated to diabetes. CONCLUSIONS/INTERPRETATION: These data confirm the importance of RD, including persistent microalbuminuria, as a marker of mortality risk and suggest that type 1 diabetes patients without renal disease achieve long-term survival comparable to the general population.

Cells in bone marrow and in T cell colonies grown from bone marrow can suppress generation of cytotoxic T lymphocytes directed against their self antigens.

Both normal mouse bone marrow and cells from T cell-containing colonies grown in vitro from normal bone marrow contain cells which can specifically suppress the development of cytotoxic T lymphocytes capable of recognizing alloantigens on the bone marrow or colony cells. Suppression, as assessed by reduction in cytotoxic activity, is produced by adding bone marrow or colony cells to mixed lymphocyte reactions between lymph node responder cells and irradiated histoincompatible spleen stimulator cells. The cytotoxic activity is reduced if the added bone marrow or colony cells are syngeneic or semisyngeneic to the stimulator cells but not if they are allogeneic. Suppression results from a reduction in the number of cytotoxic lymphocyte precursor cells activated in the cultures. The suppressor cells in bone marrow are radiation sensitive and Thy-1 negative; those in colonies grown from bone marrow are radiation resistant and Thy-1 positive.

Differentiation from precursors in athymic nude mouse bone marrow of unusual spontaneously cytolytic cells showing anti-self-H-2 specificity and bearing T cell markers.

We describe an in vitro limiting dilution culture system that supports growth and differentiation of nylon wool nonadherent bone marrow cells from athymic nude mice. Cells were seeded at low cell numbers (5-120 cells per 20-microliter microculture well) in the absence of added filler or feeder cells but in the presence of conditioned medium. Microwells positive for growth appeared to contain a single clone of cells that adhered together to form a tight cluster referred to here as a colony. A fraction of colonies contained cells that expressed an unusual spontaneous cytolytic activity. They lysed syngeneic or semisyngeneic Con A blast or tumor cell targets but seldom lysed H-2-incompatible Con A blast or tumor target cells, even in a lectin-facilitated assay. A large fraction of colonies contained lymphoid cells that expressed the T cell markers Thy-1 and Lyt-1. Colonies expressing spontaneous cytolytic activity and also containing cells with Thy-1+ and/or Lyt-1+ markers could be grown from nylon wool nonadherent nude marrow cells depleted rigorously by cell sorting of cells expressing either of these markers. Expression of Thy-1 and spontaneous cytolytic activity in a particular colony was significantly correlated. Short-term lines established from cytolytic colonies with T cell markers maintained both characteristics. The cytolytic effector cells observed in these cultures may represent an early stage in the development of the T cell repertoire.

Inhibition of the immune response by 7S antibody mechanism and site of action.

A cell culture system was used to investigate the mechanism of action of the feedback inhibition caused by specific 7S antibody. It was found that preincubation of spleen cells with specific 7S antibody led to a marked reduction in the in vitro response of the treated spleen cells to the antigen used to prepare the antibody. The inhibition was not caused by a carry-over of free antibody nor by the release of 7S antibody from the cells. Rather, the preincubation appeared to specifically inactivate one of the cells required for initiation of an in vitro response. Since the suppression could be reversed by addition of untreated cells, it was possible to characterize the properties of the reconstituting cell. This cell is identified as the nonlymphoid accessory cell (A cell) by several criteria. (a) Suppression can be demonstrated only in assay systems requiring functional A cells. (b) The most active sources for reconstitution are also good sources for A cells. (c) The sedimentation velocity of the reconstituting cell is identical with that for A cells. (d) Like A cells, the reconstituting cell is resistant to high doses of ionizing radiation. (e) The reconstituting ability is not affected by anti-theta antibody. Of the three cells required for the initiation of an immune response, A cells, bone marrow-derived cells, and thymus-derived cells, the data are only compatible with the reconstituting cell being an A cell. Additional experiments suggest that the Fc portion of 7S antibody binds to the surface of A cells. Thus, fluorescein isothiocyanate-labeled 7S antibody binds specifically to cells with properties similar to those described above, and F(ab')(2) fragments, lacking Fc portion, are unable to cause immunosuppression when they are preincubated with spleen cells. It is possible that this binding is related to the specific suppression caused by 7S antibody molecules.

Restriction of specificity in the precursors of bone marrow-associated lymphocytes.

Previous work has shown that the immediate precursor of B lymphocytes (PB cell) has many properties that distinguish it from both B lymphoctes and hemopoietic stem cells. Size, density, tissue distribution, and sensitivity to cytotoxic antisera differ for each type of cell. The work described here was designed to study three aspects of the differentiation of PB cells. First, since PB cells probably have immunoglobulin surface receptors, fluorescein-labeled anti-immunoglobulin antiserum was used in an attempt to investigate directly the physical properties of PB cells. The use of this labeled antiserum revealed a population of cells with properties similar to the PB cells defined by the functional assays. Second, the differentiative potential of PB cells was studied by comparing the size of the total population of PB cells, as determined with fluorescein-labeled anti-immunoglobulin antiserum, to the size of the population of PB cells responding in a functional assay with a specific antigen. The cells responding in the functional assay represent only 0.1% of the total population of PB cells. This observation suggests that PB cells are not pluripotent stem cells of the immune system. Finally, the kinetics of the differentiation of PB cells to B lymphocytes was studied. The differentiation to mature lymphocytes involves at least one intermediate stage in which cells larger than mature B cells are active in a functional assay for B cells. These large B cells are present in irradiated mice soon after transplantation of PB cells, but by 20 days the majority of the B cells are typical small lymphocytes.

Correlation between lymphocyte-induced donor-specific tolerance and donor cell recirculation.

Intravenous infusion of mice with major histocompatibility complex (MHC) incompatible lymphocytes can inhibit the response of recipient T cells capable of recognizing the injected cells, and can enhance survival of grafts sharing MHC with the injected cells. However, neither T cell inactivation nor graft survival enhancement is always achieved. This is particularly true for donor cells that are fully allogeneic (as compared to semiallogeneic) to the recipient. We show here that both donor-specific induced response reduction and graft survival enhancement are directly correlated with the ability of the injected lymphoid cells to persist in the recirculating lymphocyte pool of the host. Whether donor cells persist correlates inversely with the level of natural killer cell (NK) activity in the host. Fully allogeneic cells can only persist in hosts with low NK activity and can then induce response reduction. Both persistence and response reduction are abrogated by injection of the host with poly-I:C, a treatment that boosts host NK activity. The same treatment also destroys the ability of semiallogeneic injected cells to persist, to induce response reduction, and to enhance skin graft survival.

In vivo administration of histoincompatible lymphocytes leads to rapid functional deletion of cytotoxic T lymphocyte precursors.

It is well established that a single intravenous injection of F1 lymphocytes can rapidly and specifically reduce the ability of a parental recipient to generate CTL against donor alloantigens in a subsequent MLR. By fluorescently labeling the injected cells, we have been able to identify, and if desired, remove them in cell suspensions prepared from recipient spleen and lymph node. The injected cells, whether F1 or syngeneic, appeared to form part of the normal recirculating pool. Removal of injected F1 cells from responder lymph node or spleen cell suspensions had no effect on the response reduction observed in the 5-d in vitro MLR (typically 80% reduction for responder cells taken 2 d after injection of F1 cells). When the frequency of CTL precursors (CTLp) was measured by limiting dilution, it was reduced to the same degree as the MLR response, implying that response reduction is due to a reduction in the number of activatable CTL in the responder cell suspension. An equal mixture of responder cells from treated (i.e., F1 injected) and control mice gave a measured CTLp frequency equivalent to the average of the separate frequencies, implying the absence of suppressor cells active in vitro. Labeled F1 cells recovered from a first recipient could be used to induce response reduction in a second recipient. The results are discussed in terms of APCs that functionally delete rather than stimulate CTLp that recognize them (i.e., a "veto mechanism"). These experiments appear to rule out a role for in vivo-induced suppressor cells up to 8 d after injection of semiallogeneic cells but do not address the question of whether they are induced at later times.

Characterization of apoptosis-resistant antigen-specific T cells in vivo.

Clonal deletion via activation-induced apoptosis (AIA) of antigen-specific T cells (ASTC) plays a very important role in the induction of peripheral tolerance. However, none of the studies performed so far has shown a complete deletion of ASTC, a small population always persisting in the periphery. The mechanism by which this small population of ASTC escapes AIA has not been determined. Since the existence of these ASTC may influence the outcome of autoimmune diseases and long-term graft survival, we have characterized the properties of these residual ASTC in vivo with the objective of determining mechanisms that may contribute to their persistence. It was found that the resistance of the residual ASTC to AIA is not due to lack of activation or Fas/Fas-L expression. Compared to those susceptible to AIA, the residual ASTC express a high level of Th2-type cytokines that may help them to escape from AIA. Furthermore, they are able to suppress proliferation of other ASTC, suggesting they may, in fact, prolong tolerance in vivo.

A quantitative assay for the progenitors of bone marrow-associated lymphocytes.

A cell transfer assay system was developed to study the precursors of bone marrow-associated (B) lymphocytes in the adult mouse. The rationale of the assay is to inject into irradiated mice a cell suspension depleted of B lymphocytes, to wait a period of time to let precursor cells differentiate to B lymphocytes, then to correlate the number of B cells present in the recipient mice with the number of precursor cells injected. The assay as described was shown to be linear in the range of 10(5)-3 x 10(6) fractionated bone marrow cells. Kinetic studies indicated that precursor cells start producing detectable numbers of B cells within 3 days after transplantation; B cell activity then increases with a doubling time of 24 hr. Physical characterization of that precursor cell has shown that it is lighter and sediments faster than small lymphocytes. Precursor cells were found in bone marrow and spleen but could not be detected in peripheral lymph nodes. Results of physical analysis also indicate that the precursors of B lymphocytes described here may not be pluripotent stem cells for the immune system.

Quantitative studies on the precursors of cytotoxic lymphocytes. III. The lineage of memory cells.

In the course of a mixed lymphocyte culture, memory cells are produced which can give rise to a large secondary cytotoxic lymphocyte response on reexposure to the sensitizing alloantigen. We have studied the lineage of these memory cells using a clonal assay for precursors of cytotoxic T lymphocytes (CLP). Our data provide conclusive evidence that individual CLP, upon stimulation with alloantigens, gives rise to clones which contain memory cells of the same specificity as the CLP. Only half of the clones that responded in the primary stimulation could be reactivated upon exposure to the original priming alloantigen.

The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems.

The precise relationship between the stem cells for the lymphoid system and those for the blood-forming system is unclear. While it is generally assumed that the hemopoietic stem cell, the spleen colony-forming unit (CFU-S), is also the stem cell for the lymphoid system, there is little evidence for this hypothesis. To investigate the stem cells in these two systems, we irradiated bone marrow cells to induce unique chromosome aberrations in the stem cell population and injected them at limiting dilution into stem cell-deficient recipients. Several months (between 3 and 11) were allowed for the injected cells to repopulate the hemopoietic system. At that time, the bone marrow, spleen, and thymus were examined for a high frequency of cells having the same unique chromosome aberration. The presence of such markers shows that the marker was induced in a cell with extensive proliferative capacity, i.e., a stem cell. In addition, the splenic lymphocytes were stimulated with phytohemagglutinin (PHA) or lipopolysaccharide (LPS) to search for unique chromosomes in dividing T and B cells, respectively. Finally, bone marrow cells were injected into secondary irradiated recipients to determine if the marker occurred in CFU-S and to determine whether or not the same tissue distributions of marked cells could be propogated by bone marrow cells in a second recipient. After examination of 28 primary recipients, it was possible to identify three unique patterns of stem cell regeneration. In one set of mice, a unique chromosome marker was observed in CFU-S and in PHA- and LPS-stimulated cultures. These mice provide direct evidence for a pluripotent stem cell in bone marrow. In addition, two restricted stem cells were identified by this analysis. In three recipients, abnormal karyotypes were found only in myeloid cells and not in B and T lymphocytes. These mice presumably received a marked stem cell restricted to differentiate only into myeloid progeny. In three other recipients, chromosome aberrations were found only in PHA-stimulated cells; CFU-S and cells from LPS cultures did not have cells with the unique chromosome. This pattern suggests that bone marrow contains cells committed to differentiation only into T lymphocytes. For each of the three types of stem cells, secondary recipients had the same cellular distribution of marked cells as the primary recipients. This observation provides further evidence that unique markers can be induced in both pluripotent and restricted stem cells.

Functional heterogeneity in allospecific cytotoxic T lymphocyte clones. I. CTL clones express strong anti-self suppressive activity.

Five out of five allo-specific cytotoxic T lymphocyte (CTL) clones tested strongly suppressed the development of CTLs directed against the H-2 haplotype of the CTL clone and independent of the H-2 specificity recognized by the CTL clone. This was shown by including 100-1,000 cells from the five clones in one way mixed lymphocyte reaction (MLR) cultures in which the stimulator cells were of the same H-2 type as the CTL cells. When these cultures were assayed for cytotoxicity against the stimulator cell haplotype, the cytotoxic activity was decreased in a CTL cell dose-dependent manner by 50 to more than 90%. Suppression was usually not observed in MLR cultures where the CTL-H-2 type was identical with the responder cells or was different from both the responder or stimulator cells. Suppression was demonstrated not to be due to "cold" target inhibition at the time of cytotoxicity assay. Even if the added CTL were completely removed after 48-72 h of culture, significant suppression was obtained. Suppressive ability did not appear to be correlated with the level of allo-specific cytotoxic activity present in the CTL clones, but might involve direct killing of MLR precursor cells by cells in the added CTL clones. The suppression observed here, which is anti-self from the point of view of the added CTL clone, appears to be triggered by precursor cells in the MLR responder population recognizing MHC determinants on cells from the added CTL clone. This peculiar type of suppression, in which the regulator regulates on being recognized, has been christened the veto phenomenon and may play a role in maintenance of self tolerance.

Quantitative studies on the precursors of cytotoxic lymphocytes. VI. Second signal requirements of specifically activated precursors isolated 12 h after stimulation.

It is shown that, in a mixed lymphocyte reaction, the production of cytotoxic T lymphocytes (CL) from cytotoxic T lymphocyte precursors (CLP) requires two signals which are separated time. Using a flow cytometer-cell sorter and a vital, fluorescent DNA stain, Hoechst 3342, CLP specific for the stimulator cells can be separated from other CLP and from stimulator cells 12 h after initiation of mixed lymphocyte cultures. These CLP are in a state of partial activation and can produce CL in the absence of stimulator cells if a second signal in the form of a concanavalin A-induced spleen cell supernate factor is added. Specific CL are also generated when the partially activated CLP are cultured with both nude spleen cells and stimulator cells. In this case it appears that an interaction between the stimulator cells and the nude spleen cells leads to production of the second signal.

In vivo requirement for a radiation-resistant cells in the immune response to sheep erythrocytes.

Experiments have been done to establish whether the radiation-resistant or A cell has a specific function in the initiation of an immune response in mice to sheep erythrocytes (SRBC). All previous demonstrations using accessory (A) cells have involved in vitro assays and are possibly explainable as tissue culture artifacts. If A cells are essential, it should be possible to demonstrate their requirement in vivo. Therefore we first established such conditions. Two methods were found for creating an A-cell deficiency in vivo: (a) A cells disappear gradually from the spleens of irradiated mice, presumably by migration since A-cell function was shown not to be decreased by irradiation. If 3 days elapse between irradiation and transplantation of mixtures of bone marrow and thymus cells (which provide B and T but few A cells), the usual synergistic response does not occur. Addition of large numbers of freshly irradiated spleen cells to the mixture of bone marrow and thymus completely restores the immune response. (b) Injection of 10(10) horse erythrocytes into mice suppresses A-cell activity in these mice 24 hr later; a much reduced response to SRBC is obtained when they are given at this time. The response can be partially restored if irradiated spleen cells are given with the SRBC. This observation formed the basis for a quantitative in vivo assay for A cells in which the magnitude of restoration by various suspensions of irradiated cells was used to estimate the A-cell activity of that suspension. A quantitative in vitro assay for A cells was also developed. It was essential for this assay that the total cell number, B-cell number, and T-cell number be kept constant and that only the number of A cells be allowed to vary. Only under these conditions was the response a linear function of the number of A cells added. If the in vivo and in vitro assays are detecting the same class of radiation-resistant cells, the physical properties of the cells active in each assay should be identical. Spleen cells were separated on the basis of both density and sedimentation velocity. Fractions from both separation methods were tested for their content of A cells using both the in vivo and in vitro assays. The density and sedimentation profiles of A cells were similar in both assays. The demonstration that a radiation-resistant cell is required in vivo and that this cell has properties identical to the radiation-resistant cell required in vitro indicates that this cell (the A cell) is directly involved in the initiation of an immune response to erythrocyte antigens.

Requirement for non-T cells in the generation of cytotoxic T lymphocytes in vitro. I. Use of nude mice as source of non-T cells.

The ability of small numbers of LN cells to produce cytotoxic lymphocytes on in vitro culture with allogeneic stimulator cells is greatly augmented by the addition of spleen cells from athymic nude mice. The possibility that the synergism is a result of improved culture conditions or a "feeder effect" is excluded. All cytotoxic cells found in these cultures are shown to be T cells and to arise from precursors contained in the LN-cell component. The nude spleen cell component appears to be providing a required non-T cell which has been lost from the LN component through dilution. Synergism between the two components can occur whether they are syngeneic or allogeneic provided that both can recognize the same alloantigens in the stimulator population.

Mitogenic analysis of murine B-cell heterogeneity.

The B-cell mitogens lipopolysaccharide (LPS), Nocardia water-soluble mitogen (NWSM), and dextran sulfate (DxS) react with different subpopulations of B lymphocytes. Selective in vitro killing of cells responding to either LPS or NWSM has little effect on the in vitro response to the other mitogen, although the response to DxS is reduced in both cases. If, after selective in vitro killing, cells are injected into irradiated mice for 2-3 wk before measuring their in vitro mitogen responses, the same specificity pattern is seen. Thus, one is dealing with different B-cell subpopulations rather than different stages of maturation of a single population. Treatment with various alloantisera and complement before measuring the mitogen response to LPS and NWSM shows that (a) whereas all LPS response cells carry surface Ig, a subpopulation of NWSM responsive cells does not; (b) both LPS- and NWSM-responsive cells carry I-A antigens but might not I-E or I-J antigens; (c) all LPS-responsive cells carry I-C antigens, whereas approximately 25% of NWSM responsive cells do not: (d) there is a subpopulation of NWSM-responsive cells carrying neither surface Ig nor I-C antigens and resistant to anti-theta treatment.