The Lyt phenotype of cells involved in the cytotoxic response to syngeneic tumor and of tumor-specific suppressor cells

Suppressor cells, which specifically suppress the in vitro response of syngeneic spleen cells to the DBA/2 mastocytoma, P815, were identified in the spleens of DBA/2 mice injected intraperitoneally with membrane extracts of the P815 tumor. The Lyt phenotypes of various effector cells were determined. DBA/2 allogeneic killer cells were identified as Lyt-¹²⁺, whereas the syngeneic effector cells were found to be predominantly Lyt-²⁺. The suppressor cell population lost its ability to suppress the in vitro cytotoxic anti-P815 response after treatment with anti-Lyt-1 serum plus complement but not after treatment with anti-Lyt-2 serum, indicating that an Lyt-¹⁺ cell is essential in this suppression.     

Leukemia L 1210 cells induce depletion of Lyt 2+ thymocytes

The thymocytes were analyzed on the 7th day after i.p. inoculation of 10(6) leukemia L 1210 cells to syngeneic DBA/2 Wf mice. A three-fold decrease of the total number of thymocytes was found as well as 1.7-fold decrease of the per cent of thymocytes with Lyt 2+ phenotype, while the per cent of cells with phenotypes Thy 1.2+ and Lyt 1+ was unchanged.     

Defective T helper activity in the spleen of BALB/c mice immune to a syngeneic fibrosarcoma

Summary BALB/c mice were immunized with the syngeneic 3-methylcholanthrene-induced fibrosarcoma CA-2 by the growth and excision method. When lymphoid cells from different organs of these tumor-free mice were tested in a direct ⁵¹Cr-release assay, peritoneal exudate cells but not spleen cells displayed specific cytotoxicity against the syngeneic tumor target. A cytotoxic response could be obtained by tumor-immune spleen cells when cultured in a mixed lymphocyte tumor cell culture (MLTC) at high but not low density although at the same effector/stimulator ratio. Lack of cytotoxic activity in low density MLTC was not due to an impairment of cytotoxic precursors since cytotoxicity was rescued by adding exogenous interleukin-2 in experimental conditions in which no lymphokine-activated killer cells could develop relevant anti-CA-2 lysis. When low density MLTC were supplemented with either 800 R-irradiated cells or nonirradiated, negatively selected Lyt 1⁺ cells from the same immune mice, induction of a cytotoxic response against CA-2 occurred and interleukin-2 production became detectable. Additional studies indicated that spleen cells of CA-2-immune mice were also impaired in their ability to provide help to syngeneic thymocytes for the generation of cytotoxic T lymphocytes against C57BL/6J alloantigens. Dilution effect of helper cells due to immunization procedures was excluded since spleen cells of mice immunized against another BALB/c tumor, the YC8 lymphoma, or against DBA/2 minor histocompatibility antigens provided good help to thymocytes against the same alloantigens. These results indicate that tumor-immune animals may also have selective T helper defects in an important lymphoid organ like spleen.     

Thymic lymphocytes: II. Phenotypic modifications of thymocytes after concanavalin A stimulation in the presence of interleukin 2: Early modifications of Lyt 1+2+ subset and later proliferation of cells with more mature phenotypes

Cortical thymocytes are devoid of any immune function, as tested by presently available techniques. The ability of this subpopulation to respond to mitogens or antigens in the presence of interleukin 2 (IL-2) produced by activated mature T lymphocytes has been claimed but is still questioned. In an attempt to study the participation of the different thymocyte subsets and especially that of the cortical type, phenotypic modifications were examined during concanavalin A activation in the presence of IL-2. An immunofluorescent double labeling technique with anti-Lyt 1 and anti-Lyt 2 antibodies was used which led to the determination of four different phenotypes: Lyt 1⁺2⁺, Lyt 1⁺2^?, Lyt 1^?2⁺, and Lyt 1^?2^?. Careful analysis of cell viability in culture and expression of the results in absolute numbers of living cells per culture allowed us to follow modifications of small cellular subsets. Cultures of total thymocytes and PNA-agglutinated (enriched in Lyt 1⁺2⁺ cells) and non-PNA-agglutinated cells (enriched in Lyt 1⁺2^?, Lyt 1^?2⁺, and Lyt 1^?2^? cells) were studied. It was shown that thymocyte activation began by early phenotypic modifications which took place within the first 2 hr of culture but only when Con A plus IL-2 were used. These modifications imply the reduction of the Lyt 1⁺2⁺ pool and a compensatory enhancement of Lyt 1^?2⁺ and Lyt 1^?2^? cells, without modification of the total cell number or [³H]thymidine incorporation. These early phenotypic changes are interpreted as the modulation of antigens on the surface of Lyt 1⁺2⁺ cells. The second phase of thymocyte activation implies cell death (essentially Lyt 1⁺2⁺ cells) and cell proliferation. The cells which specifically proliferate in the presence of Con A and IL-2 are Lyt 1⁺2^? and Lyt 1^?2⁺, the latter always being present in greater number. Cell survival and absolute number of Lyt 1⁺2^? and Lyt 1^?2⁺ cells in the activated PNA^?-enriched population are always higher than in total thymocyte and PNA⁺ cells cultures. Thus, if Lyt 1⁺2⁺ cortical thymocytes do not proliferate by themselves, they seem to intervene by providing Lyt 1^?2⁺ cells which proliferate secondarily.     

The Lyt phenotype of the T cells in an antitumor adoptive transfer differs for “parent to F1” and “parent to parent” combinations

The T-cell subset mediating tumor graft neutralization was characterized in a methylcholanthrene (MC) tumor system. Lyt 1⁺ cells were critical for the successful prevention of outgrowth of the tumor cells in graft neutralization assays with irradiated recipients. Elimination of Lyt 1⁺ cells abolished the outgrowth inhibitory effect exerted by T-cell-enriched populations derived from syngeneic or semisyngeneic mice immunized with the H-2-carrying MC-induced M-A tumor. In accordance, lymphocyte populations containing 98% Lyt 1⁺ cells derived from M-A-immune mice, mediated a complete transplantation immunity against this tumor. When the immune T cells admixed to the tumor inoculum were syngeneic to the recipient (i.e., A-derived cells were transferred to A recipients, or F₁ to F₁), elimination of the Lyt 2⁺ cells did not influence the potential to inhibit tumor outgrowth. The presence of Lyt 1⁺2^? cells were thus necessary, and sufficient, in the syngenic combination. A reduction of the graft-inhibiting potential occurred after elimination of Lyt 2⁺ cells from the A-derived M-A immune T-cell population when the recipients were semisyngeneic (i.e., (A/Sn X A.SW)F₁, (A/Sn X CBA)F₁, or (A/Sn X C57Bl/6)F₁). Consequently, only in the semisyngeneic, but not in the syngeneic combinations, was the transfer of Lyt 2⁺ cells necessary for optimal graft inhibition. It can be concluded that the genotypic relation between the donor and the recipient influences the prerequisites of the tumor cell neutralization.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: IV. Correlation with H-2 haplotypes

In vitro primary syngeneic sensitization on monolayers of thyroid epithelial cells was performed with 21 inbred strains of mice representing 11 original H-2 haplotypes. Significant differences in the proliferative responses, assessed by thymidine uptake, were found to be related to the major histocompatibility complex haplotype. This result was further confirmed using congenic resistant strains of mice. In comparison with the experimental autoimmune thyroiditis induced by syngeneic thyroglobulin and adjuvant, primary syngeneic sensitization on monolayers of thyroid epithelial cells appeared to be under the same genetic control (H-2^k strains being good responders, while H-2^b mice are poor responders).     

Adoptive transfer of experimental allergic encephalomyelitis in SJL/J mice after in vitro activation of lymph node cells by myelin basic protein: requirement for Lyt 1+ 2- T lymphocytes

Optimal conditions were established for the adoptive transfer of experimental allergic encephalomyelitis (EAE) in SJL/J mice. Lymph node cells from SJL/J mice primed in vivo with myelin basic protein (BP) were incubated in vitro with BP. These cells proliferated specifically to BP and when transferred at the optimal conditions into syngeneic mice induced EAE in 100% of the recipients. The in vitro proliferative response to BP was dependent on the presence of Lyt 1+ 2- T lymphocytes. Furthermore, when the activated LNC were treated before transfer with anti-Thy 1 or anti-Lyt 1 antibody and C, neither clinical nor histologic signs were observed in the recipients, whereas treatment with anti-Lyt 2 antibody and C had no effect. These results indicated that Lyt 1+ 2- T cells are responsible for the transfer of EAE.     

Functional maturation of mouse CD4+CD8- thymocytes induced by medullary-type thymus epithelial cells

Murine CD4⁺CD8^- (CD4SP) thymocyte subset is a heterogeneous population, in which the Qa-2^- cells are less functional, whereas the Qa-2⁺ cells are fully functional. Evidence is provided here that the transition from Qa-2^- to Qa-2⁺ CD4SP thymocytes is an intrathymic process of differentiation induced by thymic medullary-type epithelial cells. The separated Qa-2^-CD4SP could be induced to express Qa-2 molecules up to 84%- 89% of the total viable celb after cocultured for 3d with MTEC1 cells, a murine thymic medullary type epithelial cell line established in our laboratory. Kinetic study showed that both the percentage of Qa-2⁺ cells and the density of the expressed Qa-2 molecules on CD4SP thymocytes induced by MTEC1 were progressively increasing in 72-h cultures. The MTECl-induced Qa-2⁺CD4SP thymocytes were fully functional, which exhibited capabilities of proliferation and cytokine secretion in response to Con A stimulation as high as those of freshly isolated Qa-2⁺CD4SP thymocytes. The profile of cytokines secreted by MTECl-induced Qa-2⁺CD4SP thymocytes was Thy 0 type specified by the production of IL-2, IL-4 and IL-6. The results suggest that Qa-2^-CD4SP thymocytes may give rise to the Qa-2⁺CD4SP thymocytes, and acquire fully functional competence in thymic medulla under the foster of local epithelial cells.     

Profiling calcium signals of in vitro polarized human effector CD4+ T cells

Differentiation of naïve CD4⁺ T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca²⁺ signals, mainly mediated by the store operated Ca²⁺ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4⁺ T cell subsets show differential Ca²⁺ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4⁺ effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca²⁺ release activated Ca²⁺ currents (I_CRAC) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca²⁺ profiles of helper CD4⁺ Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4⁺ T cells.     

Plasmodium chabaudi AS: Distinct CD4+CD25+Foxp3+ regulatory T cell responses during infection in DBA/2 and BALB/c mice

Malaria infections display variation patterns of clinical course and outcome. Although CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells play an essential role in immune homeostasis, the immune regulatory roles involved in malaria infection remains to be elucidated. Herein, we compared the disparity in Treg cells response during the course of blood stage Plasmodium chabaudi chabaudi AS (P. c chabaudi AS) infection in DBA/2 and BALB/c mice. BALB/c mice initiated a Th1/Th2 profile respond to P. c chabaudi AS infection, but DBA/2 mice failed to control P. c chabaudi AS infection and almost of them died post-peak parasitemia. At the peak parasitemia, we found that higher proportion of Treg cells with elevated Foxp3 expression in DBA/2 than in BALB/c mice. We used anti-CD25 mAb to deplete Treg cells and found that the survival time and rate were prolonged in DBA/2 mice treated with anti-CD25 mAb. Treatment with anti-CD25 mAb in vivo led to enhanced pro-inflammation responses and Foxp3 expression decline on Treg cells. In contrast, after DBA/2 was treatment with anti-IL-10R mAb, IL-10R blockade in vivo caused excessive pro-inflammation responses and Foxp3 expression loss on CD4⁺CD25⁺ T cells. Earlier death was found in all of DBA/2 mice with anti-IL-10R mAb. It suggested that IL-2 and IL-10 signal involved in maintaining Foxp3 expression on Treg cells. In all, the moderate suppressive activity of Treg cells may facilitate resistance to P. c chabaudi AS infection.     

Characterization of a murine thymic CD4+ T cell subset-TCRαβ+ 3G11-6C10- CD4+ CD8-thymocytes

The presence of a relatively mature CD4⁺ CD8⁻ (SP) T cell subset in mouse thymus has been demonstrated. Composing of 10% of total CD4SP thymocytes, this subset is defined by the absence of 3G11 and 6C10 expression with a phenotype of CD69^+/−, HSA^med/lo and heterogeneous for Qa-2 expression. The proliferation capability of TCRαβ⁺ 3Gl l⁻ 6C10⁻ CD4⁺ CD8⁻ thymocytes was high while using Con A stimulus. And Con A stimulation could result in secretion of IL4, IL-10, IL-6 and a little amount of IFNγ. IL-2 was barely detectable. This is distinct from typical Th0 type cytokines. The cells of this subset were NK1.1 negative, but strongly expressed GATA-3 mRNA. The results suggest that the CD4⁺ subset of 3G11⁻ 6C10⁻ NK1.1⁻ phenotype possesses immunocompetent cells with functions characteristic of Th2-like cytokines, which may indicate the cells at transitional status from Th0 to Th2, with a propensity to Th2. Project supported by the National Natural Science Foundation of China (Grant No. 39730410).     

Cloning of caf1 +, caf2 + and caf4? + from Schizosaccharomyces pombe : their involvement in multidrug resistance, UV and pH sensitivity

We previously identified four nuclear genes (caf1 ⁺ to caf4 ⁺) in Schizosaccharomyces pombe, mutations in which can confer caffeine resistance. Here we report the cloning and sequencing of caf1 ⁺, caf2 ⁺ and caf4 ⁺. All three genes are allelic to genes (hba1 ⁺ , crm1 ⁺ and trr1 ⁺ , respectively) involved in multidrug resistance mechanisms or in stress response systems. In agreement with this the caffeine-resistant mutants caf1(hba1)-21, caf2(crm1)-3 and caf4(trr1)-83 are also resistant to brefeldin. Disruption of caf1(hba1) ⁺ and caf4(trr1) ⁺ makes cells sensitive to high pH. The overlapping ranges of pleiotropic effects and the genetic interaction detected between caf1(hba1) ⁺ and caf2(crm1) ⁺ suggest that the three genes function in interlinked systems. Received: 9 March 1998 / Accepted: 16 September 1998     

Expansion of tumor-specific cytolytic T lymphocytes using in vitro restimulation with tumor-specific transplantation antigen

Tumor-specific T lymphocytes (CTL) induced by in vivo immunization of C3H/HeJ mice with the syngeneic methylcholanthrene (MCA)-induced fibrosarcoma MCA-F were expanded in vitro by restimulation with 1-butanol-extracted, isoelectrophoretically purified, tumor-specific transplantation antigen (TSTA) in combination with purified rat interleukin-2 (IL-2) and fresh, syngeneic, 2000-R-irradiated, adherent splenic antigen-presenting cells (APC). The cultured immune T-cell population, containing 40–55% Lyt 2⁺ and 40–60% L3T4⁺ cells, displayed TSTA-specific proliferative and cytotoxic activities in vitro. The expanded T cells appear to recognize butanol-extracted TSTA in association with specific H-2 class I antigens, as revealed by the benefit of syngeneic over allogeneic cells as APC and by the adverse effect of depletion using anti-H-2K, but not anti-Ia, monoclonal antibodies. In adoptive transfer assays in vitro, expanded T cells specifically neutralize homotypic, but not heterotypic, tumor growth in vivo. Based upon the effects of depletion of T-lymphocyte subpopulations using monoclonal antibodies, the Lyt 2⁺ cytotoxic T lymphocytes (CTL) appear to display greater in vivo neutralizing activity than L3T4⁺ T cells. Thus in vitro stimulation of in vivo-immunized T cells, using butanol-extracted TSTA in combination with IL-2 and syngeneic APC, expands tumor-specific CTL.     

Relationship between intracellular K+ concentrations and K+ fluxes in growing and contact-inhibited cells

The K⁺ content and the K⁺ flux were measured in the cell lines ME₂ and MF₂ isolated from plasmocytoma MOPC 173. Both cell lines were shown to have the same K⁺ content and the same K⁺ steady state flux per unit of surface area.In ME₂ cells, no modification of the exchange movement was observed during contact inhibition. However, contact-inhibited cells exhibited an increased resistance to depletion, characterized by a lower K⁺ net movement.The (Na⁺ + K⁺)-ATPase measured in homogenates is poorly correlated to in vivo cation fluxes both because of the enhancement due, presumably, to the drop of K⁺ concentration on the cytoplasmic face of the membrane and because of losses during preparation which can be conspicuous, especially in contact-inhibited cells.The K⁺ net flux is considerably increased when the intracellular K⁺ level is reduced after preincubation of the cells in a K⁺-free medium. Thus, internal K⁺ seems to regulate the K⁺ influx.     

Na+-dependence of 45Ca2+ uptake in adult rat isolated cardiac cells

We developed a technique that yields isolated adult rat myocytes, 70% of which are elongated and morphologically similar to intact tissue. Electrophysiological studies showed most of these cells were quiescent, Ca²⁺-tolerant and exhibited normal action potentials accompanied by contractions. We analyzed ⁴⁵Ca²⁺ uptake data in terms of instantaneous, fast and slow compartments. 69% of total exchangeable Ca²⁺ was found in the slow compartment; the rest was almost equally divided between the instantaneous and fast compartments. Replacement of extracellular Na⁺ by Li⁺ or Tris increased ⁴⁵Ca²⁺ uptake by the fast compartment; high [K⁺]_o increased this uptake further. These increases appeared to be related also to internal concentrations of Na⁺. This conclusion was supported by experiments with digitonin-treated cells. Our results indicate that the way Na⁺-dependent ⁴⁵Ca²⁺ uptake is affected by [Na⁺]_o, [Na⁺]_i and [K⁺]_o is compatible with the Na⁺-Ca²⁺ exchange mechanism. Our preparation should prove useful in studies of regulation of Ca²⁺ transport in cardiac muscles.     

mTOR inhibitor INK128 attenuates systemic lupus erythematosus by regulating inflammation-induced CD11b+Gr1+ cells

Systemic lupus erythematosus (SLE) is an autoimmune disease, characterized by systemic chronic inflammation that can affect multiple major organ systems. Although the etiology of SLE is known to involve a variety of factors such as the environment, random factors and genetic susceptibility, the exact role of CD11b⁺Gr1⁺ myeloid cells in lupus progression is not fully understood. Myeloid-derived CD11b⁺Gr1⁺ cells are thought to be a heterogeneous group of immature myeloid cells with immune function. Some studies have reported that CD11b⁺Gr1⁺ cells and the activation of mTOR pathway are involved in the pathogenesis of systemic lupus erythematosus (SLE). However, it is still not clarified about the mechanism of influence of lupus microenvironment and mTOR signaling on CD11b⁺Gr1⁺ cells. In the present study, we found that the percentage of CD11b⁺Gr1⁺ cells increased prior to the abnormal changes of Th17, Treg, T and B cells during lupus development. TLR7 and IFN-α signaling synergized to promote CD11b⁺Gr1⁺ cell accumulation in an mTOR-dependent manner. Moreover, compared to a traditional mTOR inhibitor, INK128 inhibited more effectively the disease activity via regulating CD11b⁺Gr1⁺ cell expansion and functions. Furthermore, TLR7/IFN-α-modified CD11b⁺Gr1⁺ cells promoted unbalance of Th17/Tregs and were inclined to differentiate into macrophages via the mTOR pathway. In conclusion, CD11b⁺Gr1⁺ cells increased in the early stages of the lupus progression and mTOR pathway was critical for CD11b⁺Gr1⁺ cells in lupus development, suggesting the changes of inflammation-induced CD11b⁺Gr1⁺ cells initate lupus development. We also provide evidence for the first time that INK128, a second generation mTOR inhibitor, has a good therapeutic action on lupus development by regulating CD11b⁺Gr1⁺ cells.     

Natural regulatory T cells mediate the development of cerebral malaria by modifying the pro-inflammatory response

Cerebral malaria (CM) is a severe neurologic complication that arises predominantly in children and non-immune adults infected with Plasmodium falciparum. In the current study, the dynamics of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Tregs) and pro- and anti-inflammatory cytokines were analyzed in P. berghei ANKA (P.bANKA)-infected C57BL/6, BALB/c, and DBA/2 mice. We showed that C57BL/6 mice were susceptible to CM, while BALB/c and DBA/2 mice were resistant to CM and succumbed to hyperparasitemia and severe anemia. The proportion and absolute numbers of Tregs in BALB/c and DBA/2 mice were significantly higher than in C57BL/6 mice. The levels of pro-inflammatory cytokines, such as IFN-γ, TNF-α, IL-6, IL-17 and NO in CM-susceptible C57BL/6 mice were obviously higher than in CM-resistant BALB/c and DBA/2 mice, while the level of the anti-inflammatory cytokine IL-10 was the opposite to that of pro-inflammatory cytokines, confirming that an appropriate balance between pro- and anti-inflammatory immune responses is essential to control the pathogenesis of severe malaria, and Tregs are important regulators if this balance is to be maintained. In vivo depletion of Tregs significantly protected C57BL/6 mice from experimental CM and the production of pro- and anti-inflammatory cytokines was reversed, indicating that this cell population contributes to pathogenesis by modulating the balance of pro- and anti-inflammatory responses. Our data demonstrate that Tregs mediate the incidence and outcome of CM in P.bANKA-infected mice by modifying the pro-inflammatory response.     

Regulation of an in vitro anti-DNA antibody response by thymocytes and T cells from NZB/W and normal mice

The spontaneous in vitro anti-DNA antibody response generated by preautoimmune and many normal mouse spleen cells was suppressed by the addition of syngeneic thymocytes or splenic T cells. Suppressive activity was found in normal mice (DBA/2J) and to an equivalent degree in the autoimmune (New Zealand Black X New Zealand White)F1 (B/W) strain. The suppressor cells were cortisone-resistant, radiosensitive and carried Lyt 1 and Lyt 2 markers. Nonspecific suppression was not involved since the primary and primed in vitro anti-sheep erythrocyte (anti-SRBC) responses were unaffected. Both spontaneous and lipopolysaccharide-stimulated anti-DNA antibody responses could be suppressed. There was no difference in the suppressive activity of cells from young or old, normal or autoimmune mice. These T cells may therefore play a role in preventing the anti-DNA antibody response in normal and young B/W mice, but evidently fail to influence the development of in vivo anti-DNA autoimmune responses in the old B/W mice.     

Characterization of an in vitro proliferation response to solubilized Trichinella spiralis antigens: Role of Ia antigens and Ly-1+ T cells

An antigen extract from Trichinella spiralis muscle larvae was prepared and used to immunize strains of mice which were either relatively resistant (B10.S) or susceptible (B10.BR) to oral infection with T. spiralis larvae. Proliferation of cells from the draining lymph nodes was then measured in vitro after stimulation with the T. spiralis extract as well as appropriate control antigens. Primed cells from resistant B10.S mice responded better to challenge than did cells from the susceptible B10.BR strain. Cell-depletion experiments involving B10.S cells indicated that the in vitro cell proliferation response is dependent upon Ly-1⁺ T cells. The data were also consistent with a requirement for Ly-1⁺, -2⁺, and -3⁺ amplifier cells. Administration of anti-I^s serum to the cultures specifically inhibited (nearly 75%) the cell proliferation response. The potential applications of this system as a tool in immunogenetic analyses of immunity to T. spiralis are discussed.     

Studies on T helper cells and the specificity of their soluble products for induction of cytotoxic T cells directed against trinitrophenyl-modified self

The role of T helper cells (Th) and their soluble products in the generation of a cytotoxic T-cell (CTL) response of thymocytes to trinitrophenyl (TNP)-modified syngeneic cells was investigated. The Th have a Thy 1⁺ Lyt 1⁺2^? surface phenotype, and produce at least two soluble helper factors. Production of factors requires stimulation of primed Th by specific antigen (self-TNP), and depends on a Thy 1⁺ Lyt 1⁺2^? cell. Factors present in supernatants after 5 hr of stimulation act preferentially on antiallogeneic precursor CTL (pCTL); factors present at 24 hr act preferentially on self-TNP-specific pCTL with a variable activity for alloantigen-specific pCTL. These results are interpreted as suggesting a possible role for helper factors having selective action in generation of CTL responses.