From: Billi Goldberg <bigoldberg@igc.apc.org>
Subject: CD8s, Clonal Anergy, and Fauci
Note: Copyright 1992, Dan R. Greening. Non-commercial reproduction allowed.
Archive-Number: 58
Organization: unspecified
Date: Thu, 28 Jan 93 12:11:05 PST

The following lead statement and the Go and Otten articles were faxed by 
me on 1/23/93 to G. Pantaleo of Fauci's Lab.

What is very interesting is that amount of research being done by 
Fauci's group on cytotoxic T cells. There are, of course, those that 
will stick to CD4 counts until you know what freezes over, but the 
cutting edge of research is on CTLs, NKs, and Th1/Th2, T1/T2 and how 
they are influenced by cytokines especially lymphokines. The Fauci 
articles are critical because they show a pattern and direction of 
research.

Fauci's new article on the pathogenesis of AIDS should be published in 
NEJM on 2/4/93. The delay was caused be required revisions and 
shortening.
=======================================================================
          CD8 CELLS, CLONAL ANERGY AND FAUCI'S ARTICLES
                         by Billi Goldberg

I think that the Go et al. article helps explain some of the 
proliferative problems of CD8+ HLA-DR+ in vitro. As I have stated 
previously, when macrophages and LC/DC (professional antigen presenting 
cells which saturate the mucosal tissues) are infected there appears to 
be a costimulatory problem probably with CD28(T cell)/B7 (APC) which 
could be either contact or signal transduction. This, then, could result 
in a proliferative problem but wouldn't interfere with the lytic ability 
of CD4+ (cytotoxic)  and CD8+. Langerhans cells differentiate into 
veiled dendritic cells when migrating from the skin and then into 
interdigitating dendritic cells in the lymph nodes; this is quite 
different than follicular dendritic cells as you well know.

There seems to be little question about the HIV infection of LC/DC. It 
is quite interesting that contact sensitization results in approximately 
one-third of the cutaneous LC migrating from the skin to the nodes for 
destruction. I have not seen any studies that mentions mucosal LC 
migration, but it just might happen. These possibly infected cells would 
be destroyed in the nodes and replaced by non-infected LC. By doing this 
weekly, the co-stimulatory function of LC/DC would be preserved 
eliminating the anergy that is probably the primary cause of the 
decreased CD4s and CD8s during disease progression and inability to 
control OIs. Interesting concept that just might explain why DNCB users 
stay alive and control OIs. The Go et al. article explains how the APCs 
could be destroyed by the anergized T cell clones.

It is too bad that the old-school immunologists have such a difficult 
time accepting the professional antigen presenting capabilities of 
dendritic cells. Too many immunologists just write them off as 
specialized macrophages. Of course, that just may be so, but so what.
The only way to get systemic immune responses (takes about 5 days) in 
the nodes is with dendritic cells; macrophages only result in localized 
responses that last about 24 hours.

I think the Th1/Th2 and T1/T2 theories explain quite a bit. It is 
interesting that CD8 suppressor cells might just be nothing more than a 
lack of interferon-gamma and a surplus of IL-4. Maybe the secret might 
be to shut down Th2/T2 which interferes with Th1/T1 by the production of 
IL-10 that inhibits interferon-gamma production. Selgame et al. article 
in Science, 10/11/91:289-282 is very interesting and might be well worth 
reading again especially the part about CD8 cytotoxic and suppressor 
cells. DNCB initiates a pure Th1 cell mediated immune response.
====================================================================== 
Go C, Lancki DW, Fitch FW, Miller J. Anergized T cell clones retain 
their cytolytic ability. Journal of Immunology, January 15, 
1993;150(2):367-376.

Abstract: CD4+ T cells have been described to have both helper and lytic 
function. The helper function of Th1 cells in particular can be 
inactivated by inducing the T cell into a state of nonresponsiveness in 
which the T cell is no longer capable of producing IL-2 or proliferating 
in an autocrine way to a conventional antigenic stimulus. To determine 
whether the lytic ability of Th1 cells can also be rendered 
nonfunctional upon anergy induction, we induced Th1 clones into a 
nonresponsive state and tested their ability to lyse target cells in an 
Ag-specific and MHC class II-restricted manner. We show that cells newly 
induced into an anergic state were able to lyse target cells 
nonspecifically. This effect was short-lived and after resting in 
culture media, the cells regained their ability to lyse target cells in 
an Ag/MHC-specific manner, and this ability was comparable to normal 
resting T cells. In contrast, the helper function of these cells 
remained non-responsive, and the cells were unable to proliferate or to 
secrete IL-2 in response to the same antigenic stimulus used for lysis. 
Therefore, the lytic pathway appears to be regulated separately from the 
proliferative/lymphokine pathway(s) and is not affected long-term by an 
anergic stimulus.

Page 373: The most striking observation from these studies is that 
although anergized Th1 cells fail to secrete IL-2 and to proliferate to 
restimulation, they retain normal lytic activity. Although there is a 
slight decrease in Ag/MHC-specific cytolytic activity shortly after 
induction of anergy, after long rest periods in culture, the anergized T 
cells are able to lyse their target cells as specifically and 
efficiently as resting T cells. The retention of lytic activity by these 
cells suggest that activation of the IL-2 production pathway is 
regulated separately from activation of the lytic pathway and that lytic 
ability is not dependent on the presence of costimulation at the time of 
TCR engagement. These results are in agreement with a recent analysis of 
CD8-positive T cells, where IL-2 production, but not lytic ability, was 
sensitive to anergy induction. In contrast, neither lymphokine 
production nor lytic function of Th2 cells are sensitive to conditions 
that induce anergy in Th1 and CD8 T cells clones.
====================================================================== 
Otten GR; Germain RN. Split anergy in a CD8+ T cell: receptor-dependent 
cytolysis in the absence of interleukin-2 production. Science, 1991 Mar 
8, 251(4998):1228-31. 

Abstract: Engagement of the antigen-specific receptor (TCR) of CD4+ 
T lymphocytes without a second (costimulatory) signal prevents the 
subsequent production of interleukin-2 (IL-2) by these cells. Because 
IL-2 is a key immunoregulatory lymphokine and is also produced by a 
subset of CD8+ T cells that are able to kill target cells, the effect of 
engaging the TCR of one such clone in the absence of costimulatory 
signals was examined. The capacity for TCR-dependent IL-2 production was 
lost, indicating comparable costimulator-dependent signaling 
requirements for IL-2 production in CD4+ and CD8+ T cells. However, 
TCR-mediated cytotoxicity was not impaired, implying that costimulation 
is required for only certain TCR-dependent effector functions. 
====================================================================== 
De Maria A; Pantaleo G; Schnittman SM; Greenhouse JJ; Baseler M; 
Orenstein JM; Fauci AS. Infection of CD8+ T lymphocytes with HIV. 
Requirement for interaction with infected CD4+ cells and induction of 
infectious virus from chronically infected CD8+ cells. Journal of 
Immunology, 1991 Apr 1, 146(7):2220-6. 

Abstract: In this study, we have investigated the basic requirements for 
HIV-1 infection of CD8+ lymphocytes in vitro. Unfractionated PBL 
obtained from healthy HIV-1 seronegative donors were activated with PHA 
and infected in vitro with HIV-1LAV. Based on immunofluorescent 
labeling, the vast majority of cells (85 to 97%) surviving peak virus 
replication belonged to the CD8+ subset and only a small percentage (0.5 
to 1.5%) were CD4+. Amplification of HIV-1 proviral sequences by 
polymerase chain reaction performed on the sorted surviving CD8+ cells 
demonstrated that CD8+ cells harbored HIV-1 proviral DNA. In addition, 
stimulation of these HIV-1-infected, CD8(+)-sorted cells either with PHA 
or anti-CD2 mAb resulted in induction of virus replication, as measured 
by reverse transcriptase activity. Electron microscopic analysis of CD8+ 
cells chronically infected with HIV-1 and stimulated with PHA showed 
typical virions budding from, and associated with, the surface of cells 
immunolabeled with gold beads directed toward the CD8 molecule. 
Infection of CD8+ cells with HIV-1 occurred only when CD4+ cells were 
present in the PHA-activated lymphocyte population exposed to HIV-1 at 
the beginning of the culture or when sorted CD8+CD4- lymphocytes were 
cocultured with autologous sorted CD8-CD4+ cells that had been 
previously infected with HIV-1. Coculture of these cells with PHA-blasts 
and incubation of their supernatants with a CD4+ cell line showed that 
these chronically infected CD8+ cells could spread HIV-1 infection to 
uninfected CD4+ cells after stimulation with PHA or anti-CD2 mAb. 
Therefore, these results suggest that the minimal requirement for in 
vitro infection of CD3+CD8+CD4- lymphocytes is the presence of infected 
CD4+ cells and that infected CD8+ T lymphocytes can further spread the 
infection to CD4+ cells. 
====================================================================== 
Pantaleo G; De Maria A; Koenig S; Butini L; Moss B; Baseler M; Lane HC; 
Fauci AS. CD8+ T lymphocytes of patients with AIDS maintain normal broad 
cytolytic function despite the loss of human immunodeficiency virus-
specific cytotoxicity. Proceedings of the National Academy of Sciences 
of the United States of America, 1990 Jun, 87(12):4818-22. 

Abstract: In this study, we have investigated the potential mechanisms 
responsible for the loss of human immunodeficiency virus type 1 
(HIV-1)-specific cytolytic activity in the advanced stages of HIV-1 
infection. We have demonstrated that HIV-1-specific cytotoxic T 
lymphocytes are predominantly contained within the CD8+DR+ subset. 
Furthermore, we have shown by a redirected killing assay that there is a 
dichotomy between HIV-1-specific cytolytic activity and broad cytolytic 
potential since the cytolytic machinery of CD8+DR+ cells is still 
functioning even in patients with AIDS who have lost their HIV-1-  
specific cytolytic activity. In addition, by comparative analysis of 
these two types of cytolytic activity over time we have demonstrated a 
progressive loss of HIV-1-specific cytolytic activity in the advanced 
stages of the disease, whereas the cytolytic potential remained 
unchanged regardless of the clinical stage. As previously shown in 
patients with AIDS, even in asymptomatic HIV-1-seropositive patients, 
CD8+DR+ cells from the same patient, compared to CD8+DR- lymphocytes, 
showed a substantial reduction in their ability to proliferate in vitro 
in response to different stimuli, such as mitogens (phytohemagglutinin 
and phorbol 12-myristate 13-acetate) and monoclonal antibodies directed 
against CD3, CD2, and CD28 molecules, and displayed a defective 
clonogenic potential. Thus, on the basis of these results we propose 
that the loss of HIV-1-specific cytolytic activity in HIV-1-infected 
individuals may result at least in part from a progressive decrease in 
the pool of HIV-1-specific cytotoxic T lymphocytes belonging to the 
CD8+DR+ subset whose ability to expand has been impaired. 
====================================================================== 
Pantaleo G; Koenig S; Baseler M; Lane HC; Fauci AS. Defective clonogenic 
potential of CD8+ T lymphocytes in patients with AIDS. Expansion in vivo 
of a nonclonogenic CD3+CD8+DR+CD25- T cell population. Journal of 
Immunology, 1990 Mar 1, 144(5):1696-704. 

Abstract: This study examines the potential mechanism(s) responsible for 
the defective clonability of CD8+ T lymphocytes in patients with AIDS. 
By the combined use of one- and two-color fluorescence cytofluorometry 
we have shown an increase in the number of circulating DR+ cells due to 
the expression of DR on a relatively large proportion of T lymphocytes 
(one-third of CD3+ cells), the majority of them belonging to the CD8+ 
subset. In addition, the majority of CD8+DR+ cells in AIDS patients did 
not express CD25 Ag (the receptor for IL-2), a surface marker generally 
expressed on normal activated T lymphocytes. Sorted CD8+DR+ and 
CD8+DR- cell populations were analyzed comparatively for their ability 
to proliferate in response to different stimuli, including anti-CD3, 
anti-CD2, alone or in combination with anti-CD28 mAb and mitogens such 
as PHA, alone or in combination with PMA. We have demonstrated that 
CD8+DR+ cells were severely defective in their proliferative response to 
triggering via these major pathways of T cell activation even when an 
exogenous source of IL-2 or IL-4 was added to the microcultures 24 h 
after initiating the cultures. In contrast, CD8+DR- cells showed a 
significant proliferation in response to the different stimuli and the 
proliferative response was strongly enhanced by the addition of IL-2 or 
IL-4. At the end of the stimulation period CD8+DR+ and CD8+DR- 
proliferating populations were analyzed for CD25 Ag expression. Only 1 
to 10% of CD8+DR+ cells expressed CD25 antigen compared with 40 to 50% 
of CD8+DR- cells. The proliferative defect of CD8+DR+ cells was further 
confirmed in experiments performed at the clonal level. The analysis of 
the frequency of proliferating T lymphocyte-precursors in both CD8+DR+ 
and CD8+DR- subsets showed that the defective clonogenic potential of 
CD8+ cells in AIDS patients could be in large part ascribed to CD8+DR+ 
cells. Five percent of CD8+DR+ cells showed a clonogenic potential 
compared to the 25% of CD8+DR- cells. Finally, we analyzed the surface 
expression of VLA-2 Ag, a marker of a chronic state of T cell 
activation, on circulating T lymphocytes. We have shown that a large 
proportion of CD3+DR+CD25- cells (50 to 80% in the different patients 
with AIDS analyzed) expressed VLA-2 Ag.(ABSTRACT TRUNCATED AT 400 WORDS) 
====================================================================== 
Koenig S; Earl P; Powell D; Pantaleo G; Merli S; Moss B; Fauci AS. 
Group-specific, major histocompatibility complex class I-restricted 
cytotoxic responses to human immunodeficiency virus 1 (HIV-1) envelope 
proteins by cloned peripheral blood T cells from an HIV-1-infected 
individual. Proceedings of the National Academy of Sciences of the 
United States of America, 1988 Nov, 85(22):8638-42. 

Abstract: Freshly separated unfractionated peripheral blood mononuclear 
cells (PBMC) and cloned cell lines from a healthy human immunodeficiency 
virus 1 (HIV-1)-seropositive individual were examined for cytotoxic 
responses to HIV proteins expressed by recombinant vaccinia viruses. It 
was found that freshly isolated PBMC recognize variant envelope proteins 
of HIV-1 but not a more distantly related envelope protein derived from 
the simian immunodeficiency virus (SIVmac). Although the effector cells 
were predominantly CD8+, both MHC-matched and -unmatched target cells 
were lysed. Cytotoxic T lymphocyte (CTL) clones were found to lyse cells 
expressing HIV-1 envelope or reverse transcriptase. In contrast to the 
cytotoxic response detected with PBMC, the cloned CTLs were major 
histocompatibility complex (MHC) class I restricted. Our finding that a 
cloned CTL line lysed cells expressing highly divergent HIV envelopes 
strongly suggested that a conserved epitope was recognized. 
Identification of these shared epitopes may assist in designing a 
vaccine for HIV-1 that could stimulate MHC-restricted cytotoxic 
responses. 

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