The effects of aromatic and aliphatic maleimide crosslinkers on anti-CD5 ricin immunotoxins

The aromatic maleimide crosslinkers m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), sulfosuccinimidyl 4-(p-maleimidophenyl) butyrate (S-SMPB) and m-maleimidobenzoylsulfosuccinimide ester (S-MBS) and the aliphatic crosslinker N-gamma-maleimidobutyryloxysuccinimide ester (GMBS) were used to make anti-CD5 intact ricin immunotoxins (IT). IT made with the various crosslinkers were compared under standard conjugation conditions for differences in yield, toxicity of the toxin moiety, binding of the antibody moiety, IT activity, and IT specificity. Our findings showed that IT yield was dramatically improved using crosslinkers with an aromatic, rather than an aliphatic configuration. Gel analysis showed that all IT were of similar, but not identical composition. Conjugation resulted in several IT species including antibody linked to one or two molecules of ricin. For MBS IT and S-SMPB IT, differences in amounts of IT in final fractions and IT in fractions after removal of IT species containing galactose binding sites showed that differences in yield may be attributable to the formation of IT species with obstructed galactose binding sites. All IT bound selectively by FACS analysis and blocking studies. The aliphatic GMBS crosslinker yielded the most toxic IT in cell-free translation assays as well as in shorter-term protein synthesis inhibition and mitogen assays. However, evaluation in the longer-term, more sensitive clonogenic assay showed that at 1000 ng/ml there were no differences in potency between any of the IT. We conclude that the yield of intact ricin IT can be improved using aromatic maleimide crosslinkers without sacrificing IT potency.     

T cell depletion of bone marrow for clinical marrow allografting: optimalization of conditions for depletion by anti-CD2 and anti-CD8 monoclonal antibodies with rabbit complement, and for detection of residual T cell content

Normal bone marrow obtained at harvest for bone marrow transplantation was passed over a Ficoll density gradient to obtain the mononuclear cell fraction. These cells were incubated with anti-T cell monoclonal antibodies (MAb) anti-HuLym-1 and anti-HuLym-8 plus rabbit complement, washed, and the degree of T cell depletion was assessed by culture in phytohemagglutinin, by flow cytometry and by limiting dilution analysis. The optimal protocol for T cell depletion was found to be a 2-stage incubation with 2 cycles of complement treatment, using a bone marrow cell concentration as low as possible. The precise conditions of complement dilution and incubation temperature depended on the batch of complement used, and had to be assessed for each batch. Of the 3 methods used for assessing T cell contamination, culture in PHA was found to be inappropriate due to large variation in background proliferation of treated and untreated bone marrow cells. Flow cytometry was accurate for residual T cell content of greater than 1% (corresponding to a 90-95% depletion of T cells) but could not detect lower levels of T cell contamination. The limit dilution assay was found to be by far the most sensitive, being capable of detecting a T cell contamination of 1 in 10,000 cells. When combined with flow cytometry on untreated marrow for calculation of the cloning efficiency, it gave very accurate determinations of residual T cell content of marrow.     

Selective in vivo depletion of CD4(+) T lymphocytes with anti-CD4 monoclonal antibody during acute infection of calves with Anaplasma marginale

To investigate the in vivo role of CD4(+) T lymphocytes during acute anaplasmosis, thymectomized calves were selectively depleted of CD4(+) T lymphocytes by treatment with anti-CD4 monoclonal antibody (MAb) and were then infected with the Florida strain of Anaplasma marginale in two sequential experiments (experiments 1 and 2). Treatment of thymectomized calves with a total of 5.0 mg of anti-CD4 MAb/kg of body weight during the 1st week followed by 0.3-mg/kg doses administered twice weekly for 7 weeks resulted in significant depletion of CD3(+) CD4(+) and CD4(+) CD45R(+) (naive) T lymphocytes from blood, spleen, and peripheral lymph nodes for the duration of the 8-week study, compared to the results for thymectomized control calves treated with a subclass-matched MAb. All calves became parasitemic and pyretic following experimental infection with A. marginale, and decreases in packed cell volume (PCV) coincided with peak parasitemia. No significant differences in PCV or parasitemia were observed between treatment groups. Thymectomized calves treated with anti-CD4 MAb were able to mount an anti-A. marginale antibody response, although in experiment 2, anti-CD4 MAb-treated calves had four- to sixfold lower immunoglobulin G1 (IgG1) and no detectable IgG2 anti-A. marginale major surface protein 2-specific antibody titers compared to thymectomized control calves treated with a subclass-matched MAb. At the level of CD4(+)-T-lymphocyte depletion achieved and experimental anaplasmosis induced, thymectomized anti-CD4 MAb-treated calves were able to control acute anaplasmosis. This was in contrast to the prediction that significant depletion of CD4(+) T lymphocytes would abrogate resistance to acute infection.     

Bacterial meningitis: T cell activation and immunoregulatory CD4+ T cell subset alteration.

Meningitis is the most important cause of acquired postnatal deafness and neurologic disorders in children. To determine if cell-mediated immunity is casually related to the pathogenesis of bacterial meningitis, T cell subsets were quantitated from blood of the 29 children with clinical and bacteriologic diagnosis of Haemophilus influenzae, Streptococcus pneumoniae, and Neisseria meningitidis bacterial meningitis. The CD4+ T cells increased and CD8+ T cells decreased in patients with meningitis as compared to patient control subjects (bacterial infections without meningitis) and normal healthy control subjects. An elevated percentage of CD25+ (interleukin-2 receptors) and HLA-DR+ (immune-response gene-associated antigen) T cells were detected from all patients with meningitis. All 29 patients with meningitis had highly elevated CD4+ CD45R+ (suppressor-inducer) cells and reciprocally depressed CD4+ CDw29+ (helper-inducer) cells compared with healthy age-matched normal and patient control subjects. These findings indicate characteristic immunologic T cell abnormalities from meningitis. The abnormal increase in the CD4+ CD45R+ suppressor-inducer or "virgin" cells and expression of activation antigens on T cells may be of help in future understanding of abnormal immune reactions from bacterial meningitis. However, deficiency of the CD4+ CDw29+ helper-inducer or "memory" cells may contribute to the impaired helper function for B cell-induced protective antibody synthesis to bacterial capsular polysaccharides found in this disease.     

Selective depletion of a thymocyte subset in vitro with an immunomodulatory photosensitizer

Conventional photodynamic therapy (PDT) utilizes light-absorbing compounds that have anti-cancer activity upon visible light irradiation. PDT has also been utilized for the treatment of certain immune conditions. To further understand the action of PDT upon immune cells, DBA/2 mouse thymocytes were treated with the photosensitizer benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) and/or an apoptosis-inducing anti-Fas (APO-1, CD95) monoclonal antibody. Nanomolar levels of BPD-MA in combination with nonthermal visible light irradiation rapidly induced apoptosis as gauged by DNA fragmentation assays. Thymocytes were modestly more sensitive to PDT-induced apoptosis than mature splenic T cells. BPD-MA and light or the anti-Fas antibody decreased CD4(+)CD8(+) cell numbers while relatively sparing CD4(-)CD8(-), CD4(+)CD8(-), and CD4(-)CD8(+) thymocytes. In combination, anti-Fas antibody and PDT augmented activity levels of the apoptosis-related protease caspase-3, cleavage of the caspase-3 substrate poly(ADP) polymerase, and the proportion of cells exhibiting DNA fragmentation and further impacted CD4(+)CD8(+) thymocyte survival. Although CD4(+)CD8(+) thymocytes had the greatest sensitivity to photodynamic depletion, BPD-MA was taken up by the other major thymocyte subsets with equal or greater avidity. Since CD4(+)CD8(+) thymocytes are selectively impacted by PDT and anti-Fas antibody can act in concert with PDT to further cytotoxicity, thymocytes may be useful for the identification of factors that govern immune cell susceptibility to this form of phototherapy.     

The anti-CD2 monoclonal antibody BTI-322 generates unresponsiveness by activation-associated T cell depletion

The antihuman CD2 MoAb BTI-322 (Lo-CD2a) effectively inhibits T cell responses in vitro to allogeneic cells, which is followed by unresponsiveness to the original stimulator in secondary stimulation. We studied the xenogeneic human antiporcine mixed lymphocyte reaction (MLR), and utilized anti-T cell receptor (TCR) Vbeta family antibody-induced cell proliferation to determine the specificity and mechanism. BTI-322 and its humanized version, MEDI-507, effectively inhibited the primary xenogeneic MLR. After suboptimal primary stimulation using lower numbers of xenogeneic stimulator cells, the unresponsiveness in secondary culture was apparent only for xenogeneic stimulator cells of the original SLA haplotype, and not for third-party stimulators or allogeneic cells. The inhibition of primary MLR was not observed for nylon-wool-purified T cells, but was seen after reconstitution of purified T cells with monocytes. Similarly, anti-Vbeta family-specific stimulation showed family-specific unresponsiveness in secondary culture. This required the presence of the whole BTI-322 molecule: a F(ab')2 fragment was not effective. T cells of a distinct Vbeta family were depleted after stimulation with an anti-Vbeta family-specific antibody and BTI-322. We conclude that the inhibition by BTI-322 of a primary xenogeneic MLR or the response to an anti-TCR Vbeta antibody is associated with unresponsiveness upon restimulation, due to activation-associated cell depletion. In this process, the interaction between monocytes and the Fc part of the antibody is involved. This unique characteristic of BTI-322 suggests the potential of the antibody for tolerance induction in vivo, besides the potential use as a T cell depleting agent.     

Selective depletion of the OKT 4+ 4B4+ subset in lymph nodes from HIV+ patients

We have used 4B4 and 2H4 monoclonal antibodies in conjunction with OKT 4 to quantify T cell subsets in lymph node suspensions from human immunodeficiency virus (HIV) positive subjects with lymphadenopathy syndrome. The data indicate that the reduced OKT 4:OKT 8 ratio was due to a depletion of the OKT 4+ 4B4+ subset. In contrast, there were no differences compared to reactive controls, considering the OKT8+ subpopulation. These alterations may be related to the immunological deficiency associated with HIV infection.     

T cell depletion using anti-CD2 coated magnetic beads simplifies the detection of peripheral blood plasma cells.

The detection of peripheral blood plasma cells is clinically important, but difficult to perform by use of routine smears. To simplify the detection process, the peripheral blood T cells from ten patients with known active multiple myeloma were depleted using anti-CD2 coated magnetic beads. In all cases, there was enrichment of the immunoglobulin (Ig) positive cells after T cell depletion (mean enrichment factor, 3.4; median, 3.2; range, 1.2-5.1) with a mean pre-bead %Ig+ cells of 7.3 compared to 20.4 for the post-bead sample (p = 0.005). The monoclonal plasma cells were similarly enriched and more easily enumerated on the microscope slides prepared from the T cell depleted sample.     

F(ab')2 anti-CD4 and intact anti-CD4 monoclonal antibodies inhibit the accumulation of CD4+ T cells, CD8+ T cells, and B cells in the kidneys of lupus-prone NZB/NZW mice

Murine lupus in NZB/NZW (B/W) mice is characterized by immune-complex glomerulonephritis and lymphocytic infiltration of several organs, including the kidney. We recently showed that treatment of B/W mice with F(ab')2 anti-CD4 monoclonal antibody retards autoimmunity by inhibiting the function of CD4+ cells and not by depleting them. To determine if treatment with F(ab')2 anti-CD4 prevented lymphocytic infiltration of kidneys or simply inhibited the function of the infiltrating lymphocytes, long-term survivors of treatment with F(ab')2 anti-CD4 and intact anti-CD4 were sacrificed for immunohistochemical analysis of their kidneys. Untreated B/W mice had large lymphocytic aggregates under the surface epithelium of the renal calyces. Most of these lymphocytes were CD4+ T cells, but CD8+ T cells and B cells were also present. In contrast, treatment with either intact anti-CD4 or F(ab')2 anti-CD4 substantially reduced, and in many cases prevented, the development of renal infiltrates. Treatment with either form of anti-CD4 not only inhibited renal infiltration by CD4+ T cells, but also prevented the accumulation of CD8+ T cells and B cells. These observations suggest a role for the CD4+ T cell in the accumulation of lymphocytes in target organs.     

T8 cell suppression of antigen- and mitogen-induced T4 cell dependent immunoglobulin production.

The suppressor effect of T8 cells on antigen-induced, as compared to pokeweed mitogen-induced, T4 cell dependent immunoglobulin (Ig) production by B cells of healthy subjects was studied. The antigens used were purified protein derivative of tuberculin (PPD) and tetanus toxoid (TT). The suppressor effect of T8 cells on IgG, IgM and IgA responses in co-cultures of T4 cells and B cells was significantly stronger in the pokeweed mitogen driven system than in PPD- and TT-driven cultures under the same experimental conditions.     

Comparison of anti-CD3 and anti-CD28-coated beads with soluble anti-CD3 for expanding human T cells: Differing impact on CD8 T cell phenotype and responsiveness to restimulation

Background  

The ability to expand virus- or tumor-specific T cells without damaging their functional capabilities is critical for success adoptive transfer immunotherapy of patients with opportunistic infection or tumor. Careful comparisons can help identify expansion methods better suited for particular clinical settings and identify recurrent deficiencies requiring new innovation.     

Evaluation of functional heterogeneity in the CD8 subset with T cells from T cell receptor-transgenic mice

The question of functional differentiation within the CD8 subset has been addressed in a model of TcR-transgenic (TcR-tg) mice expressing a TcR specific for H-2K^b (Ti). CD8⁺ Ti⁺ T cells present in the periphery of these mice have no cytotoxic T lymphocyte (CTL) activity unless they are stimulated with H-2K^b-expressing cells. In contrast to T cells from normal H-2^k littermates, alloantigen induction of CTL from TcR-tg mice is independent of CD4⁺ T helper (T_h) cells and is accompanied by high level secretion of interleukin-(IL)-2 by Ti⁺ CD8⁺ T cells. Precursor frequency analysis performed on CD8⁺ cells from TcR-tg mice revealed a high frequency of T_h as compared to CTL precursors. This raised the possibility of the existence of distinct subpopulations within CD8⁺ precursors with different requirements for differentiation to functional CTL. FACS analyses (performed on resting and on in vitro stimulated T cells from normal and TcR-tg mice) demonstrated a heterogeneous expression of Ly-6C on CD8⁺ cells with a large enrichment of Ly-6C^? cells among the Ti⁺ cells which persisted after stimulation with H-2^b cells in conditions that led to a homogeneous expression of the activation markers pgp-1 and CD69. The possibility that Ly-6C expression could mark functionally different subpopulations in CD8⁺ T cells was investigated. Stimulation of sorted populations of Ly-6C^? and Ly-6C⁺ cells allowed detection of CTL precursors in both these subsets and the majority of limiting dilution wells containing one pCTL also scored positive for IL-2 secretion. Thus, for CD8⁺ T cells expressing the same TcR, differentiation led to acquisition of both IL-2 secretion and CTL function and there was no evidence for the existence of a distinct population of helper-dependent CTL precursors.     

肾移植后外周血CD4+及CD8+T细胞亚群计数的临床意义

背景：临床上常以流式细胞检测受者外周血CD4、CD8细胞比值来揭示与排斥或感染相关的关系。 目的：探讨肾移植后排斥或感染时外周血CD4⁺及CD8⁺T细胞(简称CD4和CD8细胞)亚群计数的变化和意义。 方法：应用流式细胞仪检测肾移植121例受者CD4、CD8细胞数进行检测。根据入院病情将患者分为移植后正常组、急性排斥组、肺部感染组进行观察。 结果与结论：移植后正常患者和急性排斥患者相比,CD4、CD8细胞数差异均无显著性意义(P > 0.05)。肾移植后肺部感染患者CD4、CD8细胞数则均显著低于移植后正常组(P < 0.001)。当感染控制、症状改善时,CD4、CD8细胞数显著升高 (P < 0.001)。说明肾移植后CD4和CD8细胞计数可以作为免疫状态的参考,其对于感染的参考价值大于排斥,动态观察分析有助于指导治疗。     

Regulation of T cell proliferation by anti-CD49d and anti-CD29 monoclonal antibodies.

The beta 1 integrin VLA-4 (alpha 4 beta 1, CD49d/CD29), which is expressed on a large subpopulation of peripheral blood T lymphocytes, functions as a receptor for the endothelial adhesion protein VCAM-1 and the extracellular matrix protein fibronectin. Previous studies showed that immobilized fibronectin enhanced anti-CD3 monoclonal antibody (mAb)-induced T cell proliferation through binding to the integrins VLA-4 and VLA-5 (alpha 5 beta 1, CD49e/CD29). We studied the ability of the anti-CD49d mAb L25 to potentiate proliferation. T cell proliferation was induced by subthreshold concentrations of anti-CD3 mAb (mAb OKT3) coimmobilized with mAb L25 but not with coimmobilized anti-CD29 (beta 1) mAb. Soluble anti-CD29 mAb inhibited the proliferation induced by coimmobilized mAb OKT3 and L25 but not proliferation induced by mAb OKT3 with PMA or coimmobilized anti-CD26 mAb.     

The effect of T cell subset depletion on autoimmune thyroiditis in the Buffalo strain rat

The aim of these experiments was to analyse the functional roles of phenotypically defined T cells from Buffalo strain rats with immunisation or neonatal thymectomy-induced autoimmune thyroiditis. Rats were depleted either of CD8-positive T cells by administration of the Ox8 monoclonal antibody or of activated T cells by administration of low-dose cyclosporin A (Cs A) with an anti-IL-2 receptor monoclonal antibody (ART-18), and the effects on subsequent disease assessed. Even though animals were not completely depleted of Ox8 cells, immunisation-induced thyroiditis was enhanced by Ox8 treatment, whereas thyroglobulin antibodies were reduced compared with controls. Subtherapeutic doses of either Cs A or ART-18 alone had little effect on thymectomy-induced thyroiditis, in contrast to Cs A and ART-18 in conjunction, which prevented disease developing. These results suggest important roles for CD8-positive and IL-2 receptor-bearing T cells in experimental autoimmune thyroiditis.     

Selective CD4+ T cell deletion after specific activation in HIV-infected individuals; protection by anti-CD28 monoclonal antibodies

AIDS is characterized by a progressive decline in the number of CD4⁺ T cells. This is preceded by an early selective defect in the proliferation of these cells to recall antigens [1–3], pokeweed mitogen (PWM) [4–6] and to superantigens (SAg) [4,7]. In contrast, the proliferative response to phytohaemagglutinin (PHA) remains intact [1,2,5]. We and others have shown that the proliferative defect in response to some stimuli was in fact due to the induction of cell death [4,7]. The activation-induced cell death mechanism that explains the proliferative defects observed in vitro might also account for the progressive in vivo deletion of CD4⁺ T cells. Indeed, studies performed on different models of primates have shown that induction of cell death in CD4⁺ T cells was detected only when T cells were isolated from animals infected with a type of retrovirus that induces an AIDS-like disease [8]. This correlation prompted us to analyse further the mechanism of HIV-induced activation cell death to determine the specificity and rate of induction of cell death. T cells from HIV-infected individuals were activated with superantigens and the Vβ T cell receptor (TCR) expression analysed. Data presented here show that cell death is restricted to activated CD4⁺ T cells, and does not affect bystander cells. More importantly, addition of anti-CD28 MoAb specifically inhibited the induction of apoptosis, raising possibilities for therapy.     

Signals involved in T cell activation. T cell proliferation induced through the synergistic action of anti-CD28 and anti-CD2 monoclonal antibodies

Monoclonal antibodies (mAb) directed against the T cell differentiation antigen CD28 (Tp44) induce proliferation of resting T lymphocytes in the presence of phorbol esters. Moreover, it has been reported that such antibodies augment and sustain T cell proliferation induced by soluble antigens, phytohemagglutinin and anti-CD3 mAb. Recently, we have shown that in monocyte-depleted T cell suspensions, anti-CD28 mAb 9.3 and Kolt-2 were able to circumvent the requirement for interleukin 2(IL2) in T cell proliferation induced by soluble anti-CD3 antibodies. Apart from the synergy of anti-CD28 antibodies with phorbol myristate acetate and anti-CD3 antibodies, we found that anti-CD28 mAb were able to induce T cell mitogenesis in combination with an E rosette-blocking anti-CD2 antibody. In this report, we show that antibodies directed against different epitopes on the CD2 antigen can synergize with anti-CD28 mAb. Furthermore, we demonstrate that proliferation induced through the synergistic action of anti-CD28 mAb with anti-CD2 antibodies can be induced in the absence of accessory cells and is accompanied by the production of IL2 and the expression of IL2 receptors. We were unable to induce detectable Ca2+ mobilization through the simultaneous binding of anti-CD28 and anti-CD2 mAb. Taken together, these data show that IL2-dependent proliferation can be induced through the simultaneous binding of anti-CD28 and anti-CD2 antibodies, possibly through phosphatidyl inositol-independent pathways. The observations may provide further insight into the activation mechanisms of human T cells.