Limiting Dilution System Research Articles

Evaluation of Autoreactive Responses.

Loss of tolerance to self-antigens is considered to be one of the initial reasons for the onset of rheumatoid arthritis (RA). Identification of self-antigens and evaluation of autoreactive antibodies can foster understanding of the pathogenesis of the disease and the development of new therapeutics. By detection of responses to a particular self-antigen, such as α-enolase, keratin, fibrinogen, fibronectin, collagen, or vimentin, in patient- and animal model-derived samples, high-affinity T-cell receptor-dependent activation of autoreactive T cells to self-antigens can be elucidated. This chapter introduces a simple method to estimate T-cell-autoreactive responses to type II collagen (CII) in a murine collagen-induced arthritis model. A limiting dilution system is established in order to assess CII-dependent T-cell responses, which are reflected by the level of cytokine release.

Evaluation of Autoreactive Responses.

Loss of tolerance to self-antigens is considered to be one of the initial reasons for the onset of rheumatoid arthritis (RA). Identification of self-antigens and evaluation of autoreactive antibodies can foster understanding of the pathogenesis of the disease and the development of new therapeutics. By detection of responses to a particular self-antigen, such as α-enolase, keratin, fibrinogen, fibronectin, collagen, or vimentin, in patient- and animal model-derived samples, high-affinity T-cell receptor-dependent activation of autoreactive T cells to self-antigens can be elucidated. This chapter introduces a simple method to estimate T-cell autoreactive responses to CII in a murine CIA model. A limiting dilution system is established in order to assess CII-dependent T-cell responses, which are reflected by the level of cytokine release.

Frequency analysis of recipient-reactive helper and cytotoxic T lymphocyte precursors using a combined single limiting dilution assay

In the present study, in order to simplify the procedure of helper T lymphocyte precursor (HTLp) and cytotoxic T lymphocyte precursor (CTlp) assays, reduce the sample size required and compare the sensitivity of HTLp and CTLp assays in the detection of allocreative responses, the same set of limiting dilution mixed lymphocyte cultures was used and evaluated for both HTLp and CTLp frequencies. Ninety-four normal volunteer or patient/donor pairs were tested. High frequencies of HTLp (1:385 to 1:28 571) and CTLp (1:554 to 1:25 000) were detected for HLA unmatched unrelated individuals. The range of HTLp and CTLp frequencies of HLA identical siblings was 1:67 000 to 1:1 000 000 and 1:3700 to 1:1 000 000, respectively. In HLA matched unrelated pairs bot HTLp and CTLp frequencies ranged from 1:1000 to 1:500 000, overlapping the values obtained from the former two groups. High CTLp and HTLp freqencies were found in 73% and 84% of HLA matched unrelated patient/donor pairs, respetively, suggesting that the in vitro functional assays may provide additional and essential information for the selection of unrelated bone marrow donors. The results of this study support the hypothesis that the combined single limiting dilution system is an efficient and informative method to quantitatively analyse HTLp and CTLp frequecies against major and minor histoincompatibilities with the advantage of being less time-consuming, less laborious and requiring fewer lymphocytes. The results also suggest that the other HTLp assay is at least as efficient as the CTLp assay for the detection of alloreactivity other than of HLA identical siblings and may be useful for predicting graft-versus-host disease after bone marrow transplantation using HLA matched unrelated donors. The clinical value of this modified limiting dilution assay to predict graft-versus-host disease in bone marrow transplantation using HLA identical sibling donors and matched unrelated donors is being assessed.

Clonal analysis of tumor‐infiltrating lymphocytes from human primary and metastatic liver tumors

Phenotypic and functional characteristics of tumor-infiltrating lymphocytes (TIL) obtained from human primary and metastatic liver tumors were studied. Lymphocytes isolated from 18 tumors and autologous (A) peripheral blood (6 cases) were phenotyped by 2-color flow cytometry and cloned in a limiting dilution system, which allows virtually all normal T lymphocytes to proliferate; 70-80% of fresh TIL were T cells (i.e., CD3+), and the ratio of CD4+/CD8+ cells was 1.2 in both primary and metastatic liver tumors. TIL contained significantly more CD56+ (NKHI+) cells, half of which were CD3+CD56+, CD3+CD25+ cells and CD3+HLA-DR+ cells, than A-PBL. The frequencies of proliferating T-cell precursors (PTL-p) and cytolytic T-lymphocyte precursors (CTL-p) reactive with K562, allogeneic tumor cells and autologous tumor cells, were determined. Mean PTL-p frequencies for TIL from hepatocellular carcinomas, cholangiocarcinomas and metastatic liver tumors were 0.52 (0.22-0.83), 0.10 (0.05-0.16) and 0.16 (0.01-0.30), respectively. The frequency of CTL-p with natural-killer-like activity was lower in TIL than in A-PBL. The frequency of CTL-p for autologous tumor cells in fresh TIL isolated from primary liver tumors was 0.02-0.13 and 12/81 clones were reactive against autologous tumor. In contrast, only 1/66 TIL clones obtained from colon carcinomas metastatic to liver showed autotumor reactivity. No clones reactive with autologous tumor were obtained from peripheral blood of patients with liver cancer. These data indicate that substantial differences in anti-tumor functions of TIL between primary and metastatic liver tumors exist, which can be detected at a clonal level.

Resistance to murine cutaneous leishmaniasis is mediated by TH1 cells, but disease‐promoting CD4+ cells are different from TH2 cells

A limiting dilution system has been used for quantitative analysis of antigen-reactive T cells producing interleukin (IL)2, IL4 and interferon (IFN)-gamma in the course of murine infection with Leishmania major. The precursor frequencies of CD4+ cells with the potential for production of IFN-gamma, which has been associated with TH1 cells, are much higher in resistant than in susceptible mice, whereas the reverse is found for CD4+ cells secreting IL4 which have been classified as TH2 cells. Our results allow a better understanding of the relative contribution of these cell types at various stages of disease and can be summarized as follows: (a) secretion of IL4 can be demonstrated in short-term clonal cultures of CD4+ cells from L. major-infected mice, (b) CD4+ cells releasing IL2, suggested to be a characteristic of TH1 cells that predominate in resistant mice, can also be detected in susceptible mice at any time of infection, (c) both IL2 and IL4 are released by the progeny of individual T cells from susceptible mice and (d) the kinetics of precursor frequencies in genetically susceptible mice protected against the disease by prophylactic treatment are different from those of congenitally resistant mice, thus indicating that the development of lymphokine-producing T cells and the establishment of protective immunity may be regulated differently in those mice. The data suggest that resistance to disease is correlated with the presence of IFN-gamma-producing TH1 cells, while susceptibility is associated with CD4+ cells that do not segregate into the TH1 or TH2 subset but display an overlapping pattern of lymphokine activities.

Characterization of cells with T cell markers in athymic nude bone marrow and of their in vitro-derived clonal progeny. Comparison with euthymic bone marrow.

Evidence is presented that early steps in T cell differentiation, to the point of TCR expression, can occur in athymic nude bone marrow (BM) and perhaps also in normal BM. Low density nylon wool nonadherent BM from 5- to 8-wk-old nude and normal mice was found to contain dim CD3+ CD4- CD8- cells. This subpopulation was not found in scid/scid low density nylon wool nonadherent BM but was present at similar frequency in both nude and normal BM and expressed comparable fluorescence intensity. With the use of additional markers, a difference between nude and normal dim CD3+ CD4- CD8- cells was found: the ratio of J11d+/J11d- cells was at least threefold higher in nude than in normal BM. Whereas 60% of nude J11d+ CD3+ cells expressed a TCR-alpha/beta and 40% expressed a gamma/delta TCR, almost no TCR-gamma/delta expressing cells were detected in dim CD3+ cells from normal mice in either the J11d+ or J11d- subpopulation. The majority of those cells express a TCR-alpha/beta. We established that nude dim CD3+ CD4- CD8- cells were distinguishable from mature recirculating T cells and developed an in vitro clonal culture system that supported the growth of these cells. When these cells were cocultured in vitro in a limiting dilution system with inactivated anti-CD3 mAb-secreting cells, in the presence of rIL-2 and rIL-4, one in 5 x 10(2) to 1 x 10(3) cells were growth inducible. Most progeny cells expressed a CD3+ CD8+ CD4- surface phenotype. Cultures displayed nonspecific cytolytic reactivity. With the use of a sensitive dot-blot technique, transcripts coding for C alpha, C beta, VC gamma, and C delta TCR genes were detected in these cultures. Rehybridization experiments demonstrated mutually exclusive expression of either C alpha or C delta mRNA transcripts in the majority of clones selected for a high probability of clonality. Most (greater than 95%) of T cell clones derived from normal BM expressed the C alpha transcript with approximately 5% expressing the C delta transcript. In contrast, about 40% of T cell clones derived from nude BM expressed the C delta transcript whereas the remaining 60% expressed 10-fold lower C alpha message than normal BM-derived clones.

Frequencies of Borrelia burgdorferi-reactive T lymphocytes in Lyme arthritis.

Using a limiting dilution system, frequencies of Borrelia burgdorferi-reactive T cells were determined in the blood and synovial fluid of four patients with chronic Lyme arthritis (LA), one patient with acrodermatitis chronica atrophicans (ACA), two patients with other inflammatory joint diseases, and two healthy individuals. B. burgdorferi-reactive precursor T cells ranged from 1/750 to 1/8220 in case of LA and ACA patients and from 1/820 to 1/31 400 in case of controls. In vivo activated B. burgdorferi-reactive T cells were almost absent in control subjects. With one exception, they were detected in LA patients at frequencies ranging from 1/1 300 to 1/15 400. Interestingly, even after successful antibiotic therapy of LA patients, similar frequencies of in vivo activated B. burgdorferi-reactive T cells were observed in the peripheral blood, provided that low cell concentrations were used for culture. At higher cell numbers, the fraction of B. burgdorferi-reactive T cells apparently dropped, suggesting regulatory phenomena.

Autoreactive T cells in rheumatic disease (1) analysis of growth frequencies and autoreactivity of T cells in patients with rheumatoid arthritis and lyme disease

A limiting dilution system was established in order to estimate frequencies of interleukin-2 (IL-2)-responsive, autoreactive and alloreactive T cells in samples of peripheral blood (PBL) and synovial fluid lymphocytes (SFL), from patients with rheumatoid arthritis (RA) and lyme disease, as well as from healthy donors and a patient with osteoarthrosis. The frequencies of IL-2-dependent T-cell colony formation were significantly higher in patients with RA and lyme disease (median: 1 287 ) as compared to controls (median: 1 1,313 ) indicating a preactivation of T cells in these patients in vivo. Autoreactivity was measured by the proliferative response of T-cell lines to autologous irradiated PBL as stimulating cells. The frequencies of autoreactive T cells in blood were significantly higher in patients (median: 1 2,615 ) as compared to controls (median: 1 19,607 ). There was no significant difference in autoreactive T-cell frequencies between the patients' SFL (median: 1 3,185 ) and PBL (median: 1 2,615 ). In every case the frequency of alloreactive T cells exceeded the frequency of autoreactive T cells. Most autoreactive T-cell lines were also alloreactive and were shown to be MHC Class II-restricted. There is evidence of a down regulation of autoreactive T cells by suppressor cells in peripheral blood in two cases with elevated autoreactive T-cell frequencies (one RA patient and one control patient suffering from a viral infection). In contrast, no suppression of autoreactive T cells was observed in the RA patients' SFL or in PBL and SFL from patients with lyme disease. These results suggest that the chronic inflammation observed in RA and lyme disease may be supported by an elevated number of autoreactive T cells in the absence of suppressive mechanisms.

Mature T cells generated from single thymic clones are phenotypically and functionally heterogeneous.

A limiting dilution system for cloning thymic CFU (CFUt) from murine bone marrow has been critically evaluated to test the clonal origin of the thymic colonies. Simultaneous limiting dilution transfer of three populations of bone marrow, each expressing a unique allelic cell surface determinant, resulted in independent segregation of donor-derived thymocyte populations within groups of recipient mice. Statistical analysis of the data allowed an estimate of 1 CFUt/3.3 x 10(4) i.v. transferred bone marrow cells. A pulse-chase experiment was utilized to establish whether CFUt seed directly to the thymus, or whether thymic seeding is secondary to extra-thymic engraftment. The results supported the conclusion that bone marrow CFUt utilize a specific interaction with thymic blood vessel endothelial cells to recognize and enter the thymus, and that this seeding occurs within 4 h of i.v. infusion. A kinetic analysis of emigration of the CFUt progeny into the peripheral blood revealed that, in most cases, an early wave of predominantly CD4+ CD8- lymphocytes emerges from the thymus approximately 4 wk after radiation and reconstitution. In a few cases, the first progeny of CFUt to emerge from the thymus were predominantly CD4- CD8+. Commitment of CFUt to TCR beta-chain rearrangements was assessed by quantitating expression of the V beta 8 family of TCR V region genes. Although some clones expressed a significantly higher or lower percentage of V beta 8+ cells, these differences were not stable with time. Thus, CFUt do not undergo absolute commitment to cell surface phenotype of TCR rearrangement, as reflected by the phenotypes of their progeny. Clones of mature peripheral progeny of CFUt could be expanded in culture in the presence of mitogen and growth factors; approximately 30 to 50% of proliferating clones could mediate cytotoxicity in a lectin-dependent assay, further indicating that CFUt are not absolutely committed to a particular T cell function.

Determination of frequency of T cells expressing the T cell-specific serine proteinase 1 (TSP-1) reveals two types of L3T4+ T lymphocytes.

TSP-1 is a murine T cell-specific serine proteinase which is exclusively expressed in activated but not in resting T lymphocytes. Among T lymphocyte clones tested so far the enzyme was found to be associated with all Ly-2+ but only with a fraction of L3T4+ lines. Here we have applied a limiting dilution system to determine the frequency of precursor cells of resting L3T4+ and Ly-2+ lymphocytes which can be induced in vitro by antigen/lectin to express TSP-1. T cell subsets were either positively enriched by flow cytofluorometry cell sorting or by negative selection using monoclonal antibodies and complement. Following stimulation of lymphocytes in vitro, individual microwells were tested for growth by visual examination and for the TSP-1 protein/enzyme by analyzing cell lysates using either a specific rabbit anti-TSP-1 antiserum and/or the chromogenic model peptide substrate H-D-Pro-Phe-Arg-p-nitroanilide. Moreover, a large panel of L3T4+ and Ly-2+ T lymphocyte clones generated from primary cultures were similarly investigated. In some cell cultures the presence of TSP-1 was also tested on the mRNA level using a TSP-1-specific oligonucleotide probe. The data show that the majority, if not all, of antigen/lectin-induced-Ly-2+ T cells expressed TSP-1. In contrast, only 12%-27% of the growing lectin or antigen-reactive L3T4+ lymphocytes were positive for the enzyme. Studies performed in parallel with L3T4+ and Ly-2+ lymphocyte populations sensitized in bulk culture showed that under these conditions similar levels of TSP-1-specific mRNA and enzyme activity are detected in both subsets. The finding of primary L3T4+ T lymphocyte clones with distinct patterns of TSP-1 production provides evidence for the existence of two types of L3T4+ effector cells with different functional capacities. The data also suggest a cooperation between distinct L3T4+ lymphocytes for induction of optimal TSP-1 activity in L3T4+ T cells.

Enumeration of T cells reactive with Mycobacterium tuberculosis organisms and specific for the recombinant mycobacterial 64-kDa protein.

The major goal of the present study was to develop a limiting dilution system for the enumeration of T cells which respond to mycobacterial antigens. Purified T cells from M. tuberculosis-immune mice were restimulated with mycobacterial antigens and accessory cells, and after 4 days expanded with antigen, accessory cells and T cell growth factor. After another 3 days, proliferative responses were determined. Similar cultures performed without antigen served as controls. Limiting dilution analysis revealed that approximately 1/2000 to 1/3000 T cells from M. tuberculosis-immune mice responded to whole M. tuberculosis organisms while T cells from normal mice did not respond. Similar T cell numbers reacted with several mycobacterial strains indicating expression of shared T cell antigens. Using a semi-purified recombinant 64-kDa protein from M. bovis the frequency of T cells generated after immunization with M. tuberculosis which reacted with a single mycobacterial protein could be estimated. We found that approximately 1/5 of the M. tuberculosis-reactive T cells recognized this particular antigen. Immunization with the recombinant 64-kDa protein in an adjuvant containing trehalose dimycolate, monophosphoryl lipid A and mycobacterial cell wall skeleton stimulated an equally high number of M. tuberculosis-reactive T cells (1/2000). These findings demonstrate that a high proportion of tuberculosis-responsive T cells are directed against the 64-kDa protein and that immunization with this antigen in an appropriate adjuvant system is capable of stimulating high numbers of M. tuberculosis-reactive T cells. Limiting dilution analysis with a panel of mycobacterial proteins or peptides may allow their ranking from immunodominant to immunosilent and facilitate identification of antigens or epitopes relevant to protection.

Enumeration of Listeria monocytogenes-reactive L3T4 + T cells activated during infection

A limiting dilution system was developed which allows minimal estimates of the number of Listeria monocytogenes-reactive T cells from infected mice. Limiting numbers of T cells were restimulated in vitro with accessory cells in the presence or absence of antigen (heat-killed L. monocytogenes organisms) and proliferative responses determined. The responding T cells resided entirely within the L374 +, Lyt2 − (helper/inducer) T-cell subset. L. monocytogenes-reactive T cells were not demonstrable in uninfected mice nor during the first 3 or 4 days of infection. In contrast, on days 4 or 5, respectively, of infection approximately 1 1000 T cells showed a response to L. monocytogenes. Their frequency declined only slightly over the subsequent weeks and still was as high as 1 4900 4 weeks after infection when no bacteria were present in the host. The frequency of L. monocytogenes-reactive T cells depended on the number and virulence of the infecting organisms, the highest sublethal dose of virulent bacteria inducing the highest frequency. Chemotherapeutic shortening of infection between days 3 and 4 resulted in a six-fold reduction of reactive T cells. Thus, the frequency of L. monocytogenes-reactive T cells depended on the presence of bacteria in the host during the first 3 to 4 days of infection. These findings may have implications for the rational design of vaccines directed against intracellular bacterial pathogens as they raise the question whether attenuated bacterial strains of low persistence can induce sufficiently high T-cell numbers required for protective immunity.

T lymphocyte control of autoreactivity: analysis with human T cell clones and limiting dilution culture.

To investigate cellular mechanisms controlling activated autoreactive T lymphocytes, a limiting dilution system was established employing cloned autoreactive major histocompatibility complex class II specific lymphocytes (a2/7) as stimulator cells for autologous peripheral blood mononuclear cells. At low responder/stimulator ratios, cytotoxic effector cells were generated capable of lysing clone a2/7. Importantly, within the population of cells mediating autocytotoxic effector function, differential specificities were found to exist. The generation of such autocytotoxic T lymphocytes appears to be inhibited by an additional population of cells circulating at lower frequency suggesting that autoreactivity is controlled at distinct levels by discrete functional cellular subsets.

Degeneracy of H‐2 recognition by cytotoxic T lymphocytes: 10% of the total repertoire is “specific” for a given haplotype and up to 1% is self‐H‐2 reactive

The frequency of specific cytotoxic T lymphocytes (CTL) precursors in the total CTL pool was analyzed in a lectin-driven limiting dilution system. We found that up to 10% of the expressed CTL repertoire in a normal mouse is "specific" for a given allogeneic H-2 haplotype. Split-well analysis under clonal conditions demonstrates that the antigens recognized by the effector CTL are H-2 encoded. A high frequency of CTL "specific" for self-H-2 antigens was revealed in all the experiments, accounting for about 1% of the total inducible pool of CTL. These results suggest a high degree of degeneracy of H-2 recognition by CTL and the immunocompetence of self-H-2-reactive CTL precursors in normal individuals.

Human Cytotoxic T Lymphocytes

A limiting-dilution system was established to measure the frequency of alloreactive cytotoxic T-lymphocyte precursors (CTL-p) in human peripheral blood T cells. Culture medium supplemented with recombinant interleukin-2 enabled clonal expansion of all CTL-p stimulated by allogeneic peripheral blood or spleen cells. The range of CTL-p frequencies in fully HLA-mismatched responder-stimulator combinations was 1:240 to 1:1230. Split-well analysis of individual microwells showed that the cytotoxic T-cell clones generated under limiting-dilution conditions showed exquisite specificity for the stimulating alloantigens. Alloreactive CTL-p were enriched in the OKT4- T-cell subset. This limiting-dilution system was highly reproducible and can thus be applied to investigate human cytotoxic T-cell precursor frequencies in various clinically relevant situations.

Allorestricted cytotoxic T cells. Large numbers of allo-H-2Kb-restricted antihapten and antiviral cytotoxic T cell populations clonally develop in vitro from murine splenic precursor T cells.

Cytotoxic T lymphocyte (CTL) responses of splenic T cells from C57BL/6 B6) mice and mutant H-2Kbm1 (bm1) mice to haptenic (trinitrophenyl [TNP] ) and herpes simplex virus (HSV) determinants in the context of an allogenic (wild-type or mutant) H-2Kb molecule were analyzed in a modified limiting dilution system. In the B6-anti-bm1TNP mixed leukocyte reaction (MLR), estimated frequencies for precursors of CTL clones that lysed bm1TNP targets ranged from 1/120 to 1/400; in the bm1-anti-B6TNP MLR, estimated frequencies of precursors of CTL clones that lysed B6TNP targets ranged from 1/500 to 1/1,300. Estimated frequencies for precursors of CTL clones that lysed the respective unmodified and TNP-modified allogeneic targets were two- to three-fold lower. Lytic specificity patterns determined by split-well analysis showed that at least 20-30% of the generated CTL populations (selected for a high probability of clonality) in both MLR displayed allorestricted lysis of TNP-modified concanavalin A blast targets. In the B6-anti-bm1HSV MLR, estimated frequencies for precursors of CTL clones that lysed bm1HSV targets ranged from 1/70 to 1/300; in the bm1-anti-B6HSV MLR, estimated frequencies for precursors of CTL clones that lysed B6HSV targets ranged from 1/300 to 1/1,200. Again, estimated frequencies for precursors of CTL clones that lysed the respective noninfected and virus-infected allogeneic targets were two- to fourfold lower. Of the CTL populations selected for a high probability of clonality at least 30-60% displayed allorestricted lysis of virus-infected lipopolysaccharide blast targets in both MLR. It is concluded that a large fraction of clonally developing CTL populations stimulated with TNP-modified or HSV-infected allo-H-2Kb-bearing cells displayed an allorestricted pattern of recognition. It was further evident that the estimated frequencies of splenic precursors that generated allorestricted CTL clones was two- to threefold higher than the estimated frequencies of precursors that gave rise to the respective alloreactive CTL populations.

Quantitative studies on T-cell functions in [formula omitted] mice : I. Frequency analysis of precursor cells of proliferating, cytotoxic, and T-cell growth factor-secreting T lymphocytes reveals an increase in absolute numbers, with a concomitant decrease in percentage of immunocompetent cells

A limiting dilution system has been applied to compare precursor frequencies of proliferating T lymphocytes (PTL-P), of T-cell growth factor-secreting T cells (T TCGF-P), and of cytotoxic T cells (CTL-P) in lymphocyte populations of aged, MRL MP - lpr lpr (MRL- lpr) mice and the congeneic strain MRL MP -+/+ (MRL-n,), or the H-2 k-compatible strains AKR N and B10.BR responding to the mitogen concanavalin (ConA), to alloantigens (H-2) or to trinitrophenol (TNP)-modified syngeneic cells. In lymph node and spleen populations of 3- to 8-month-old MRL- lpr mice, the frequencies of H-2 d- or ConA-reactive PTL-P and T TCGF-P, and of CTL-P sensitive to either H-2 d, TNP, or ConA stimuli were between 5 to 30 times lower than in the corresponding populations of the other three strains. Furthermore, the frequencies of CTL-P progressively decreased in MRL- lpr mice from 3 to 8 months of age. In contrast, the absolute numbers of immunologically competent precursor T cells (PTL-P, T TCGF-P, CTL-P) was in general approximately 2- to 5-fold higher in MRL- lpr than in the control mice. However, these normal T cells do not seem to expand proportionally with the progressive lymphadenopathy in MRL- lpr mice since the number of T lymphocytes recovered from lymph nodes of the individual animals tested exceeded those of tissues from control mice by 30- to 300-fold. The results therefore suggest that mature T cells are progressively diluted out by abnormal lymphocytes in lymphocyte populations of aging MRL- lpr mice, thus causing a decrease of immune responses in vitro and possibly also affecting optimal cellular interactions in vivo.

Interleukin 2 enhancement of lymphokine secretion by T lymphocytes: analysis of established clones and primary limiting dilution microcultures.

The effect of exogenous interleukin 2 (IL 2) on lymphokine production by T lymphocytes was examined in two systems: the secretion of macrophage-activating factor (MAF) and interferon (IFN) by cloned long-term T cell lines, and a limiting dilution system for estimating the frequency of precursors of MAF-secreting cells in normal spleen. An IL 2-containing, MAF- and IFN-free supernatant from the EL-4 thymoma (EL-4 SN) significantly enhanced release of MAF and IFN by mitogen- or antigen-stimulated, cytolytic or noncytolytic T lymphocyte clones directed against alloantigens or Moloney leukemia virus-associated antigens. Highly purified IL 2 produced equivalent enhancement as EL-4 SN in cultures of alloreactive clones stimulated with concanavalin A. Kinetics experiments showed that EL-4 SN increased both the rate and duration of MAF release by T cell clones. EL-4 SN also increased MAF production when added during restimulation of limiting dilution cultures of positively selected Lyt-2+ and Lyt-2- C57BL/6 splenic T lymphocytes activated against DBA/2 alloantigens. This enhancement resulted in a threefold increase in the apparent precursor frequency of MAF-secreting cells among Lyt-2+ lymphocytes, but did not affect the frequency among Lyt-2- cells. Additional analysis indicated that average MAF production in cultures of Lyt-2-+ cells was sixfold lower than in cultures of Lyt-2- cells, and hence that EL-4 SN allowed detection of a significant proportion of Lyt-2+ cell cultures secreting low levels of MAF. Under these improved conditions, the MAF assay detected the majority of responding Lyt-2+ and Lyt-2- lymphocytes.

Recognition of polymorphic H-2 domains by T lymphocytes. I. Functional role of different H-2 domains for the generation of alloreactive cytotoxic T lymphocytes and determination of precursor frequencies.

In the present communication, the repertoire of alloreactive cytotoxic T lymphocytes (CTL) clones was quantitatively investigated by limiting dilution analysis and by target inhibition with a panel of monoclonal antibodies (mAb). These mAb have previously been shown to define two distinct alloantigenic domains, A and B, on the H-2Kk molecule. The Poisson distribution analysis of H-2Kk-specific CTL clones generated in a limiting dilution system revealed three CTL populations with different precursor frequencies. The high frequent population is suppressed by an unknown suppressive mechanism that allows less frequent CTL populations to become visible. Target inhibition studies with a panel of Kk-specific mAb showed that these CTL populations differ not only in their precursor frequency but also in their specificity for different H-2 epitopes on the Kk molecule. Thus clones of the high frequency population are almost exclusively specific for determinants within domain A. In contrast, the low frequency population displays predominant specificity for determinants of domain B, while the population with medium frequency is blocked equally well by mAb against either domains A or B. Each mAb blocked only a fraction of clones indicating that each CTL subpopulation may consist of a large number of clonotypes with specificity for different H-2 epitopes. The data suggest that CTL recognize basically the same polymorphic domains on the H-2Kk molecule defined by antibodies, and they show that regulatory mechanisms determine the expressed repertoire in CTL populations.

Quantitative studies on T cell diversity. I. Determination of the precursor frequencies for two types of streptococcus A-specific helper cells in nonimmune, polyclonally activated splenic T cells.

A limiting-dilution system is described that makes use of T cell growth factor T cell expansion and allows the determination of precursor frequencies for various regulatory and effector T cells in nonimmune, polyclonally, or specifically activated T cell populations. Two different sets, a frequent and a rare set, of T helper cell precursors with specificity for trinitrophenyl-group A streptococcal vaccine, could be identified: the frequent set is of the Lyt-123 phenotype, and is present at frequencies of from 1/1,000 to 1/6,000 splenic T cells. It is only active at low cell numbers, whereas it is completely inactivated at greater cell numbers, presumably by suppressor T cells of lower frequency but greater potency. The rare set is of the Lyt-1 phenotype, is present at frequencies of from 1/10,000 to 1/70,000, and is not sensitive to suppressor cells present within the tested cell numbers. We suggest that the frequent set contains primiary helper cell precursors, whereas the rare set contains helper T memory cells preselected by previous exposure to other antigens. The results are discussed with respect to other reports on the involvement of more than one set of helper cells in antibody production.