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Opioid use disorders (OUDs) are a public health concern in the United States and worldwide. Current medications for OUDs may trigger side effects and are often heavily regulated. A novel treatment strategy to be used alone or in combination with existing medications is active immunization with antiopioid vaccines, which stimulate production of opioid-specific Abs that bind to the target drug and prevent its distribution to the brain. Although antiopioid vaccines have shown promising preclinical efficacy, prior clinical evaluations of vaccines targeting stimulants indicate that efficacy is limited to a subset of subjects who achieve optimal Ab responses. We have previously reported that depletion of IL-4 with a mAb increased opioid-specific IgG2a and total IgG, and it increased the number of germinal centers and germinal center T follicular helper cells in response to antiopioid vaccines via type I IL-4 signaling. The current study further investigates the mechanisms associated with IL-4-mediated increases in efficacy and whether IL-4 depletion affects specific processes involved in germinal center formation, including affinity maturation, class switching, and plasma cell differentiation in mice. Additionally, results demonstrate that preimmunization production of IL-4 after ex vivo whole blood stimulation predicted in vivo vaccine-induced Ab titers in outbred mice. Such mechanistic studies are critical for rational design of next-generation vaccine formulations, and they support the use of IL-4 as a predictive biomarker in ongoing OUD vaccine clinical studies., (Copyright © 2023 by The American Association of Immunologists, Inc.)

A cDNA library was prepared from the murine Th cell line Th2 D.10 and used to clone the murine homologue of Ox40 by polymerase chain reaction. Comparison of the mouse sequence with the rat revealed greater than 90% homology between the two sequences at both the DNA and protein level. Northern blot analysis found that, as in the rat, Ox40 expression appears to be restricted to activated T cells. A chimeric receptor globulin was prepared to include the mouse Ox40 extracellular domain coupled to the hinge-CH2-CH3 domains of human IgG1 (Ox40-Ig). This soluble form of the molecular was then used to identify cells bearing a ligand for Ox40. FACS analysis revealed that Ox40-Ig bound to a subset of peritoneal B cells as well as to a fraction of LPS-activated splenic B cells. Immunostaining of spleen sections using an Ag-specific conjugate and Ox40-Ig found a significant proportion of antibody-forming cells co-stained with Ox40-Ig. Immunoprecipitation of cell-surface radiolabeled peritoneal B cells suggests a specific interaction with a protein of 70 kDa.

Guinea pigs were immunized with liposomal model membranes actively sensitized with 2,4-dinitrophenyl-aminocaproylphosphatidyl-ethanolamine. The immune response was characterized by the formation of both IgM and IgG anti-DNP plaque-forming cells (PFC) which paralleled the appearance of anti-DNP antibodies in the serum. Plaque inhibition by DNP-lysine indicated that the PFC produced after liposomal immunization fell in a much narrower range of avidity groups than the PFC obtained after immunization with DNP-albumin and, in this regard, resembled MOPC 315. The restricted nature of the serum IgG anti-DNP antibodies was confirmed by isoelectric focusing and revealed the expression of a limited number of clones in all cases.

The trinitrophenyl hapten (TNP) has been covalently conjugated to bacterial lipopolysaccharides (LPS) to give TNP-LPS. The site of attachment has been suggested to be in the core polysaccharide and lipid A region of the molecule and approximately 2.4 hapten molecules are bound per monomer LPS molecule. The TNP-LPS has been demonstrated to be immunogenic in vitro at very low concentration. This antigen has further been shown to initiate a T-independent TNP-PFC response. The immunogenicity of TNP-LPS is abrogated by mild alkaline hydrolysis, suggesting a requirement for intact lipid A in the initiation of an immune respose at the very low concentrations of antigen used.

Evidence is presented for two types of helper T cells in the mouse specific for a protein antigen, keyhole limpet hemocyanin (KLH). The first cell is able to help B cells respond to the hapten, trinitrophenyl (TNP), when coupled to KLH presumably through the participation of KLH-specific T cell factors. The second helper cell when stimulated with KLH is able to help B cells respond to red blood cell antigens, but not TNP-KLH, through the production of a non-antigen-specific factor. Both types of helper cells were shown to be present in the spleens of KLH-primed mice, although they occurred with different frequencies. They were shown to be different T cells by their segregation from each other in vitro at limiting dilution.

A humoral factor extracted from calf thymus (THF) restores the immunocompetence of spleen cells from neonatally thymectomized (NTx) mice to induce an in vitro graft-vs-host (GVH) response. This acquistion of immunocompetence consists of a series of biochemical events, the first of which involves an obligatory rapid increase in adenyl cyclase acitivity and in intracellular levels of cyclic AMP. Protein synthesis occurs as a further step in the events leading to induction of immunocompetence by THF and could be blocked by cycloheximide with the consequent abolishment of the immunocompetence of the lymphoid cells tested. The induction of competence by THF is accompanied by a simultaneous reduction in DNA synthesis resulting from the increase in intracellular cyclic AMP levels. These steps have been studied in the absence of antigenic stimulation which is not required for the induction of competence by THF. Spleen extracts prepared by a similar procedure as THF were found to be devoid of the properties described above.

Cultured cells from mouse thoracic duct, spleen, lymph node, Peyer's patches, and bone marrow gave rise to cells containing large amounts of cytoplasmic IgM, IgGl, and IgG2 when stimulated by bacterial lipopolysaccharide (LPS). Differntiation of IgA producers occurred in bone marrow only, and to a lesser extent than other classes. Significant IgM responses preceded development of cells containing other classes. The differentiation of IgG and IgA producers did not appear to depend on T cells, since cultures from nu/nu or thymectomized-irradiated, bone marrow-protected mice responded as well as normals. Cultures from mice rendered deficient in B cells by anti-mu treatment responded normally to T cell mitogens, but did not proliferate or give rise to immunoglobulin-secreting cells when stimulated with LPS. Bone marrow cultures gave relatively meager proliferative responses to LPS, but generated as many or more immunoglobulin-secreting cells as did other tissues.

A sequential culture technique for the in vitro induction and subsequent assay of T helper cells is employed to examine the histocompatibility requirements for antigen recognition by murine T helper cells. F1 T cells are primed in vitro with antigen-pulsed parental strain macrophages and tested for antigen-specific helper activity in cultures containing anti-Thy 1.2 serum and C treated spleen cells from hapten-primed parental or F1 mice. A semiallogenieic system is used and appropriate controls are included to avoid possible complicating effects of allogeneic interactions. The results indicate that F1 T helper cells preferentially stimulate carrier-specific anti-hapten plaque-forming cell responses in spleen cells which are H-2 identical with the macrophage used initially to prime the T cells. Parental spleen cell cultures do not respond to F1 T helper cells which were primed with the other parental strain macrophage. Supplementing this culture with macrophages which are histocompatible with those used to prime the F1T cells is sufficient to restore T helper cell activity. Thus, the genetic restriction described here is between the primed T cell and the macrophage used to elicit secondary responses and not between the T cell and B cell. The results in this semiallogeneic system, however, do not rule out the possibility of additional allogeneic genetic restrictions in the subsequent interaction of T cells with B cells.

In vitro hydrocortisone in physiologic and pharmacologically attainable concentrations caused a marked enhancement of the PWM-induced PFC response of normal human peripheral blood B lymphocytes. This effect was seen only when hydrocortisone was added within the first 24 hr of culture and only when hydrocortisone and PWM were present together in cultures. Only suprapharmacologic concentrations of hydrocortisone (10(-3) M) were capable of suppressing early B cell activation. Late stages of antibody production and secretion were resistant to suppression by even these extraordinarily high concentrations. Hydrocortisone did not replace the T cell requirement of PWM-induced PFC responses. A single dose of in vivo hydrocortisone (400 mg) to normal adult volunteers did not produce this enhancing effect when PFC responses were measured in vitro in the absence of hydrocortisone. The data strongly suggest that the enhancing effect of hydrocortisone was due not to elimination of naturally occurring suppressor cells, but to a modulation of the triggering signal either directly on the B cell itself or via the balance of positive and negative T cell regulation of B cell activation.

The primary immune response against sheep red blood cells in T cell-deficient spleen cell cultures from nude mice was tested in the absence and presence of allogeneic spleen cells. The allogeneic spleen cells differed either in regard to the major histocompatibility complex (H-2) or only with respect to the M-locus. Surprisingly the M-locus different spleen cells were almost as efficient in enhancing the anti-sheep red blood cell response in nude cultures as were the cells differing on the complete H-2 complex. Evidence is presented that AKR anti-theta serum sensitive T cells are responsible for the M-locus-dependent effect edscribed. This effect is shown to be mediated by a factor released from actived T cells stimulated in M-locus different mixed lymphocyte cultures. Since almost identical parameters have been observed in both the M-locus-dependent situation as in the "classical" allogeneic situation we concluded that an allogeneic effect can be induced by T cells responding to a complete set of the major histocompatibility complex (H-2) as well as to lymphocyte-activating determinants (M-locus) alone.

Staphylococcal enterotoxins, like several plant lectins, have been shown to stimulate mitogenic activity and lymphokine production in lymphocytes. The effect of enterotoxins A and B on the primary in vitro plaque-forming cell (PFC) response of mouse (C57BL/6) spleen cells to sheep red blood cells (SRBC) was examined. PFC responses in 4- or 5-day cultures were inhibited greater than 90% by 0.1 mug enterotoxin A and by 3- to 6-mug enterotoxin B when the toxins were added to the cultures either at the time of SRBC addition or 24 hr before. Both enterotoxins A and B were shown to be T lymphocyte mitogens. Kinetic data indicated that the enterotoxins (10 mug) inhibited the PFC response as early as day 3, but were relatively more inhibitory at days 4 and 5. The PFC response was inhibited when enterotoxins were added to cultures at the same time as antigen or a day later; it was enhanced when added at day 2; and it was unaffected when added at day 3 of 4-day cultures. Further, the presence of enterotoxin A during the first 24 hr of culture and subsequent removal was still as effective in inhibiting the PFC response as when it was present throughout the culture period. The PFC inhibitory properties of enterotoxins appear, then, to affect some early event(s) in the in vitro immune response. Alpha-methyl-D-mannopyranoside blocked the PFC inhibitory effect of concanavalin A (con A), but it had no effect on enterotoxin A. The two mitogens appear, then, to react with different receptors on the lymphocytes or to differ in the dynamics of their reactivity. The effects of staphylococcal enterotoxins on the PFC response of spleen cells were remarkably similar to those reported for lectins such as con A. Enterotoxins are structurally simpler than con A and should therefore be quite useful in studying various biologic activities of lymphocytes.

A series of experiments were undertaken in mice and rats to confirm the report that foster nursing rats of one strain, on mothers of a different strain, can influence their survival and immunologic competence as a consequence of their receiving a significant number of immunologically competent leukocytes via the milk. These experiments have included foster nursing mice and rats, either immediately after birth, or when 25 to 36 hr old, on H-2 incompatible and Ag-B compatible and incompatible mothers, respectively, and challenging them with ear skin grafts of the same genotype as their surrogate mother. They also have included fostering animals rendered tolerant of their surrogate mother's transplantation antigens. In no instance was any evidence obtained that foster nursing can prejudice the survival or influence the immunologic competence of mice or rats.

The role(s) of helper T lymphocytes in preventing or altering tolerance induction in DNP-specific B lymphocytes was studied. As DNP-reactive helper T cells were reactive against the DNP-portion of the DNP-D-GL molecule, we could probe definitively the physiological role of helper T cells in preventing tolerance induction in B lymphocytes by DNP-D-GL. The results demonstrated that the induction of DNP-specific B cell tolerance by DNP-D-GL can be completely prevented by the presence of DNP-reactive helper T cells, and provide evidence that one critical role of helper T cell participation in humoral responses to antigens is to circumvent the development of a tolerogenic signal that, in the absence of such T cell function, might otherwise ensue after binding of the antigenic determinants by specific B lymphocytes.

Rabbit antiserum to culture supernatant of concanavalin A-stimulated peripheral blood mononuclear cells was used in a reverse hemolytic plaque assay to detect activated T lymphocytes in human peripheral blood. The number of plaque-forming cells in fresh, unstimulated lymphocyte preparations was approximately 400/10(6) cells, and increased 7- to 14-fold after stimulation with a variety of mitogens and antigens. The kinetics of the increase paralleled 3H-thymidine incorporation, with a maximum on day 3 of culture. Plaques were eliminated by treatment of cells with anti-Leu-I + complement or by depletion of E-rosettes. The activated T cells were not restricted to a given inducer, suppressor, or Ia+ T cell subset, however. Both mitogen-stimulated culture supernatants and mitogen-activated lymphocytes, but not resting lymphocytes, were effective in absorbing the capacity of the rabbit antiserum to develop plaques. This suggested that the predominant specificity of this antiserum was directed toward surface membrane determinant(s) of T cells actively shed into the medium. This process was shown to require ongoing protein synthesis, and, in mitogen-stimulated lymphocyte preparations, DNA synthesis as well. This approach has enabled detection of a surprisingly large number of endogenously activated T cells in normal human blood, and should be useful for the analysis of immunoregulatory events at the single-cell level.

Studies under controlled conditions of lighting and temperature revealed clear evidence of circadian periodicity with respect to the number of PFC present in the spleens of BALB/c mice 3 or 4 days after immunization with SRBC. Striking differences in proliferative responses of spleen lymphocytes to PHA or Con A were also observed at two different circadian times. Large proliferative responses occurred at the time when injection of antigen and/or sampling for PFC yielded a low PFC formation (early in the daily dark span) and small proliferative responses occurred at the time when antigen injection and sampling yielded high formation of PFC (early in the daily light span). These findings indicate that care should be taken to control the circadian timing of stimulation and sampling in studies of immune responses, and that rhythmically varying aspects constitute a new dimension of immunologic processes awaiting further analysis in both the circadian and weekly spectral regions.

Serially transferred LAF spleen cells show a deficient 7S PFC response to TNP-Brucella when Tx as compared to normal irradiated intermediate hosts are used. This defect can be corrected by combining the spleen cells with lymph node cells from the Tx intermediate hosts. It is concluded that T helper cells, residing primarily in lymph node, are needed for efficient 19S leads to 7S conversion in the immune response.

The influence of a phase shift in the light-dark cycle on humoral immune responses against sheep red blood cells (SRBC), a thymus-dependent antigen, and against polyvinylpyrrolidone (PVP), a thymus-independent antigen, was studied by using 180 BALB/c mice and 150 C3H/HeN mice. Significant suppression of the immune response to SRBC and the number of splenic plaque-forming cells (PFC) and hemagglutination (HA) titers was observed on days 5 and 6 after inversion of the light-dark cycle. On the other hand, the number of splenic PFC and HA titers in the blood against PVP were minimally suppressed by the phase shift in C3H/HeN mice, except for distortion of the rhythmicity. Corticosterone levels in the blood on days 5 and 6 after inversion were higher than those under a normal lighting regimen. The appearance of the high corticosterone level in the blood after the inversion almost concurred with the suppression of the immune response to SRBC. A decrease of the proportion of splenic T cells was also observed on day 6 after the inversion. These results show that a phase shift in the light-dark cycle provokes suppression of the immune response to SRBC, possibly through an increase of secretion of corticosterone after light-dark inversion, which induces a decrease of the proportion of T lymphocytes in the spleen.

Previous studies have shown that rabbit antibody-forming cells in the primary and secondary response possess cell-associated antigen-binding receptors. In the present study, we demonstrate that a factor appears in the serum of rabbits following immunization which inhibits the antigen binding of up to 60% of the receptor-bearing antibody-forming cells in both the primary and secondary response. These observations were made on lymph node cells from rabbits primed with either sheep red blood cells (SRBC)3 or 3-nitro-4-hydroxy-5-iodophenylacetic acid coupled to keyhole limpet hemocyanin (NIP-KLH). The inhibitory activity is not associated with anti-SRBC or anti-NIP antibody. In the primary response to SRBC, the antigen binding by day 6 antibody-forming cells is inhibited by the autologous days 7 to 10 inactivated and absorbed serum. In the secondary response to SRBC, the inhibitory factor peaks in the serum around day 10. Later, in both the primary and secondary immune response to SRBC, the inhibitory activity of the serum decreases rapidly. In the primary response to NIP-KLH, the inhibitory activity of the immune sera increased from day 7 through day 14. The receptor-inhibiting factor is antigen specific since the serum from SRBC-primed rabbits inhibits SRBC binding by anti-SRBC antibody-forming cells, but it does not inhibit NIP binding by anti-NIP antibody-forming cells. Similarly, serum from NIP-KLH-primed rabbits inhibits NIP binding by anti-NIP antibody-forming cells, but does not inhibit the SRBC receptor on the anti-SRBC antibody-forming cells. The inhibition is not due to the presence of antihapten or anti-SRBC antibody competing with receptor sites, since the immune sera from one SRBC-primed animal inhibit antigen binding of its own antibody-forming cells, but do not inhibit the antigen binding of antibody-forming cells from other SRBC-primed rabbits.

?The stimulatory and inhibitory effects of concanavalin A (Con A) on the in vitro primary immune responses to a T-dependent antigen, sheep erythrocytes (SRBC) and a T-independent antigen, TNP-lipopolysaccharide (TNP-LPS) have been studied. Inhibition of the response to both antigens was optimal when 2 mug Con A were added at the initiation of the culture period. The response to SRBC was considerably enhanced by the addition of Con A 24 hr later. In contrast, this late addition did not stimulate the TNP-LPS response and often inhibited it. Inhibition of the TNP-LPS response required the participation of T cells since it was not observed in cells from adult thymectomized irradiated bone marrow-reconstituted (ATXBM) mice. The response to TNP-LPS was somewhat enhanced in ATXBM cells, but the degree of enhancement was strikingly less than that observed for SRBC. LPS per se did not block the stimulatory effect of Con A on the SRBC response, and was observed to act synergistically with this lectin. None of the Con A effects observed required the participation of adherent cells. These observations are consistent with a model in which different subpopulations of T cells are responsible for the inhibitory and stimulatory effects. They further suggest that the Con A inhibitory activity acts via a T cell to inhibit directly the B cell response to antigen.

B cell memory was shown to develop in congenitally athymic (nu/nu) mice after injection with small amounts of thymus-dependent antigens, in particular heterologous serum proteins, such as fown gamma-globulin (FGG) or DNP-bovine-serum albumin (DNP-BSA). Large doses of proteins (10 mg) tended to produce a specific B cell unresponsiveness, although there was still some evidence of B cell priming. The antigen did not have to be in a multivalent form to interact with B cell so as to induce immunologic memory or tolerance. In contrast to the induction of B cell memory, the production of IgG antibody in this system was found to be strongly T cell dependent. Thymus-independent antigens like LPS or POL with pronounced adjuvant effects on IgG production in normal or surgically thymectomized mice, could not replace T cells in allowing an IgG response against thymus-dependent antigens in congenitally athymic mice. However, the action of T cells once activated is likely to be non-antigen-specific, since it was shown that supernatants of antigen-activated-syngeneic T cells stimulated IgG production in cultures of primed B cell populations non-antigen-specifically.

Human peripheral lymphocytes (PBL) upon stimulation with PWM proliferate and differentiate to IgM- and IgG-producing cells. The PWM-induced Ig production in B cells was dependent on T cells, and cell-free supernatant (CFS) obtained from PWM-stimulated PBL or T cell-rich fraction replaced T cell helper functions. The active substance(s) in CFS were most likely derived from T cells. The kinetic studies showed that the proliferation of B cells took place in advance of the final differentiation to Ig-producing cells and that T cells or T cell product(s) had to exist at the initiation of cultures in order to give the maximum helper effect. However, the final differentiation of B cells to Ig-producing cells was not dependent on T cells. The helper effect of T cells or T cell product(s) on PWM-induced proliferation and differentiation of B cells was exerted across the MHC barrier. This may make it possible to apply this experimental system to the assessment of quantitative and/or qualitative changes in human helper T cells in several immunologic diseases.